CN117546000A

CN117546000A - Shredding-fixing method and shredding device for preparing biological samples

Info

Publication number: CN117546000A
Application number: CN202280044420.2A
Authority: CN
Inventors: 贾瓦德·N·阿布桑德; 莎米拉·查特吉巴塔查尔吉; A·金; 瑞恩·斯托特; J·特罗斯珀托雷斯; F·凡尔德坎纳斯
Original assignee: 10X Genomics Inc
Current assignee: 10X Genomics Inc
Priority date: 2021-06-23
Filing date: 2022-06-23
Publication date: 2024-02-09

Abstract

The present disclosure provides biological sample preparation and analysis methods that use shredding and fixation treatments in preparing dissociated fixed cells from biological tissue for use in batch assays or single cell/single cell nuclear assays, such as partition-based assays. The present disclosure also provides assay methods, including partition-based methods, that use immobilized cells prepared from biological tissue, which may optionally be used in combination with a deffixed treatment. Kits are also provided that include dissociation reagents, dissociation fixatives, and other assay reagents for use in these methods.

Description

Shredding-fixing method and shredding device for preparing biological samples

Cross Reference to Related Applications

The present invention claims the priority benefits of the following U.S. provisional application numbers: 63/214,043 submitted at month 23 of 2021, 63/213,908 submitted at month 23 of 2021, and 63/349,064 submitted at month 4 of 2022, each of which are incorporated herein by reference in their entirety.

Technical Field

The present disclosure relates generally to methods and apparatus for preparing a biological sample of fixed cells from tissue for use in batch and partition-based assays.

Background

Microfluidic techniques have been developed for introducing individual biological samples (e.g., cells) into discrete partitions (e.g., droplets). Each partition may be fluidly isolated from the other partitions such that the respective environments in each discrete partition can be precisely controlled, allowing the biological sample in each partition to be processed separately. For example, the biological sample in each zone may be bar coded and then subjected to a chemical or physical treatment, such as heating, cooling, or chemical reaction. This allows the biological sample in each discrete partition to be qualitatively or quantitatively processed in its own separate partition-based assay.

Biological samples comprising a variety of biomolecules can be processed in partition-based assays for various purposes, such as detecting disease (e.g., cancer) or genotyping (e.g., species identification). However, biological samples are unstable after removal from their viable biological niches, and their physical breakdown begins immediately. The rate and extent of dissociation is determined by a number of factors, including time, solution buffer conditions, temperature, source (e.g., certain tissues and cells have a high level of endogenous ribonuclease activity), biological stress (e.g., enzymatic tissue dissociation can activate stress response genes), and physical manipulation (e.g., pipetting, centrifugation). Degradation affects important nucleic acid molecules (e.g., RNA), proteins, and molecular complexes of higher order 3D structures, whole cells, tissues, organs, and organisms. Instability of biological samples is a significant obstacle to their use in partition-based assays (e.g., single cell assays). Sample degradation greatly limits the ability to accurately and reproducibly use such assays with a wide range of available biological samples.

The problem of instability of biological samples is complicated and diverse when using tissue samples typically comprising a plurality of cell types having different characteristics. The ability to fix and preserve the different cell types present in a tissue sample can vary greatly depending on the specific structure, function, and spatial location within the tissue of these cells. Standard biological methods for preparing samples of different cell types from tissue involve dissociating the tissue with heat and enzymes, however, these methods can greatly alter or disrupt the natural state of certain cell types. In zone-based single-cell assays, the ability to obtain accurate measurements from the different types of cells found in the tissue requires rapid immobilization and dissociation of the different cell types so that the relevant assay can be performed before sample degradation occurs. However, the use of standard preservatives and fixatives on tissue samples results in highly skewed measurements, as these standard techniques do not reach all cell types in a manner that accurately preserves their natural state in the tissue sample.

Disclosure of Invention

The present disclosure provides methods and devices for biological sample preparation and analysis that use shredding and immobilization processes in preparing dissociated immobilized cells from biological tissue for batch or single cell/single cell nuclear assays, such as partition-based assays of nucleic acid sequences present in the cells.

In at least one embodiment, the present disclosure provides a method of preparing a biological sample, wherein the method comprises:

(a) Chopping biological tissue into a tissue fragment composition;

(b) Treating the tissue fragment composition with a fixation reagent, thereby providing a fixed tissue fragment composition; and

(c) The fixed tissue fragment composition is treated with a cell dissociation agent to provide a fixed cell or fixed cell nucleus composition.

In at least one embodiment of the method of preparing a biological sample, the method further comprises: (d) The fixed cells or nuclei composition is treated with a solution comprising a unfixed agent, thereby providing multiple types of unfixed cells or nuclei from the biological tissue.

In at least one embodiment, the present disclosure also provides a method of biological tissue analysis, wherein the method comprises:

(a) Chopping biological tissue to provide a tissue fragment composition;

(b) Treating the tissue fragment composition with a solution comprising a fixative to provide a fixed tissue fragment composition;

(c) Treating the fixed tissue fragment composition with a cell dissociation agent to provide fixed cell or cell nucleus compositions, each fixed cell or cell nucleus comprising a plurality of crosslinked nucleic acid molecules; and

(d) Generating a plurality of barcoded nucleic acid molecules from the plurality of crosslinked nucleic acid molecules and a plurality of nucleic acid barcode molecules, wherein one barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules comprises i) a sequence corresponding to one crosslinked nucleic acid molecule of the plurality of crosslinked nucleic acid molecules, or a complement thereof, and ii) a barcode sequence, or a complement thereof.

(a) Chopping biological tissue to provide a tissue fragment composition;

(c) Treating the fixed tissue fragment composition with a cell dissociation agent to provide fixed cell or cell nucleus compositions, each fixed cell or cell nucleus comprising a plurality of crosslinked nucleic acid molecules;

(d) Treating the fixed cell or cell nucleus composition with a defreezing agent to provide a defreezed cell or cell nucleus composition, each defreezed cell or cell nucleus comprising a plurality of uncrosslinked nucleic acid molecules; and

(e) Generating a plurality of barcoded nucleic acid molecules from the plurality of uncrosslinked nucleic acid molecules and a plurality of nucleic acid barcode molecules, wherein one barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules comprises i) a sequence corresponding to one uncrosslinked nucleic acid molecule of the plurality of uncrosslinked nucleic acid molecules, or a complement thereof, and ii) a barcode sequence, or a complement thereof.

In at least one embodiment of these biological tissue analysis methods, the generating a plurality of barcoded nucleic acid molecules is performed in a plurality of partitions; optionally, wherein the plurality of partitions is a plurality of droplets or holes. In at least one embodiment, one of the plurality of partitions comprises a fixed cell or cell nucleus and a support comprising the plurality of nucleic acid barcode molecules; optionally, wherein the support is a bead. In at least some embodiments, the barcode sequence is a partition-specific barcode sequence.

In at least one embodiment of the biological tissue analysis method comprising treating a fixed cell or cell nucleus composition with a unfixed agent, the treatment with the unfixed agent is performed in a plurality of partitions.

In at least one embodiment of these biological sample preparation methods or biological tissue analysis methods provided by the present disclosure, the tissue fragment composition comprises particles having an average size on one side of about 500 μm or less, about 250 μm or less, about 125 μm or less, about 75 μm or less, or about 50 μm or less. In at least one embodiment of these methods, the tissue fragment composition comprises particles having an average size on one side of between about 50 μm and 500 μm, between about 125 μm and 500 μm, between about 250 μm and 500 μm, between about 50 μm and 250 μm, or between about 50 μm and 125 μm.

In at least one embodiment of these biological sample preparation methods or biological tissue analysis methods, the immobilization reagent is paraformaldehyde ("PFA"); optionally, wherein the PFA is present in the solution at a concentration of 1% to 4%.

In at least one embodiment of these biological sample preparation methods or biological tissue analysis methods, the cell dissociation agent comprises collagenase.

In at least one embodiment of these biological sample preparation methods or biological tissue analysis methods, the amount of time before the fixed tissue fragments are treated with the cell dissociation agent is at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 1 week, at least 1 month, at least 6 months, or more.

In at least one embodiment of these biological sample preparation methods or biological tissue analysis methods, the fixed cell or cell nucleus composition comprises a plurality of fixed cell types, or fixed cell nuclei from a plurality of cell types.

In at least one embodiment of these biological sample preparation methods or biological tissue analysis methods, the immobilized cells or nuclei of the immobilized cell or nucleus composition comprise a plurality of crosslinked nucleic acid molecules.

In at least one embodiment of these biological sample preparation methods or biological tissue analysis methods, wherein the method comprises treating the immobilized cells or cell nucleus composition with a destabilizing agent capable of removing crosslinks formed in the biological molecule by immobilization with 1% to 4% concentration of paraformaldehyde ("PFA") solution. In at least one embodiment, the unfixed agent comprises a compound selected from the group consisting of: compound (1), compound (2), compound (3), compound (4), compound (5), compound (6), compound (7), compound (8), compound (9), compound (10), compound (11), compound (12), compound (13), compound (14), compound (15), or a combination thereof. In at least one embodiment, the concentration of the deammobilizing agent is from about 1mM to about 500mM, from about 50mM to about 300mM, or from about 50mM to about 200mM.

In at least one embodiment of these biological sample preparation methods or biological tissue analysis methods, wherein the methods comprise treating the immobilized cells or cell nucleus compositions with a unfixed agent, the solution comprising the unfixed agent further comprises a protease. In at least one embodiment, the protease is a thermolabile protease or a cold-active protease; optionally, wherein the protease is selected from the group consisting of subtilisin a, protease K, arcticZymes protease, and combinations thereof.

In at least one embodiment of these methods of preparing a biological sample or analyzing a biological tissue, the biological tissue is selected from the group consisting of brain tissue, skin tissue, muscle tissue, smooth muscle tissue, heart muscle tissue, skeletal muscle tissue, bone marrow tissue, lung tissue, bronchus tissue, oviduct tissue, gall bladder tissue, ovary tissue, testis tissue, hypothalamus tissue, thyroid tissue, adrenal tissue, kidney tissue, pancreas tissue, small intestine tissue, large intestine tissue, colon tissue, liver tissue, lymph tissue, breast tissue, mesenteric tissue, nasal tissue, pine cone tissue, parathyroid tissue, pharyngeal tissue, laryngeal tissue, pituitary tissue, prostate tissue, saliva tissue, spinal cord tissue, spleen tissue, stomach tissue, thymus tissue, tracheal tissue, tongue tissue, urinary tract tissue, placenta tissue, artery tissue, vein tissue, and tonsil tissue.

In at least one embodiment of these biological sample preparation methods or biological tissue analysis methods, the methods may further comprise filtering and/or sieving the immobilized cells or cell nucleus compositions and/or disarming the cells or cell nuclei.

In at least one embodiment, the present disclosure provides a morcellating device for mechanically morcellating a biological tissue sample, the device comprising:

(a) A razor cartridge comprising a plurality of razor blade slots, each slot configured to insert a blade edge of a razor blade through the razor blade slot and to retain a top portion of the razor blade within the razor blade slot, wherein a bottom portion of the razor blade comprising the blade edge extends vertically below the razor cartridge;

(b) A razor blade alignment layer comprising a plurality of alignment openings, wherein each alignment opening is configured to insert a blade edge of a razor blade through the alignment layer such that a bottom portion of the razor blade comprising the razor blade edge extends below the alignment layer; and

(c) A circular sample tray holder configured to hold a sample tray containing a biological tissue sample;

wherein the razor cartridge is configured to be placed over and connected to the razor blade alignment layer such that each of the plurality of razor blade slots is aligned over each of the corresponding plurality of aligned openings; and is also provided with

Wherein the razor blade alignment layer is configured to be placed over and reversibly and rotatably connected to the circular sample disk holder such that each razor blade cutting edge extends into and toward the bottom of the circular sample disk holder.

In at least one embodiment of the shredding device, the razor cartridge includes at least two through holes and at least two screws inserted through the at least two through holes, wherein the screws are used to reversibly connect the razor cartridge to the razor blade alignment layer. In at least one embodiment, each of the at least two screws is a spring-loaded screw configured to provide a vertical lifting force to the razor cartridge relative to the alignment layer so as to provide a collapsible gap between the razor cartridge and the alignment layer. In at least one embodiment, the spring-loaded screw is configured such that the gap between the razor cartridge bottom and the alignment layer top is in the range of 0.5mm to 10mm at rest.

In at least one embodiment of the shredding device, the device further comprises a handle connected to the top of the razor cartridge; optionally, wherein the handle comprises at least one through hole for a screw to pass through in order to connect the handle to the razor cartridge.

In at least one embodiment of the shredding device: (i) The razor cartridge having a cartridge diameter, the plurality of razor blade slots being parallel and having a length of at least 85% of the cartridge diameter; and/or (ii) the razor blade alignment layer has an alignment layer diameter, the plurality of alignment openings being parallel and having a length of at least 75% of the frame diameter.

In at least one embodiment of the shredding device, the razor cartridge and razor blade alignment layer are configured such that the blade edge of each razor blade inserted into the slot and opening will contact the bottom of the sample tray when the razor cartridge and razor blade alignment layer is pushed downward.

In at least one embodiment of the shredding device, the circular sample disk holder includes a top circular rim, and the alignment layer includes an annular channel around an outer perimeter of the circular bottom surface and corresponding to the top circular rim of the sample disk holder, wherein the annular channel is configured to receive the top circular rim to reversibly and rotatably connect the alignment layer to the circular sample disk holder.

In at least one embodiment of the shredding device, the alignment layer includes a second annular channel around an outer perimeter of the circular bottom surface and within an inner circumference of the annular channel, wherein the second annular channel is configured to receive a top circular edge of a sample disk held within the circular sample disk holder.

In at least one embodiment, the present disclosure also provides a method of using a morcellating device, wherein the device includes a plurality of razor blades inserted into a plurality of razor blade slots and a plurality of aligned openings, placed over and reversibly and rotatably coupled to a circular sample disk holder containing an open circular sample disk containing a tissue sample, the method comprising:

(i) Pressing down on the razor cartridge, thereby forcing the cutting edges of the plurality of razor blades against and cutting the tissue sample contained within the sample tray;

(ii) Rotating the plurality of razor blades to a second orientation relative to the tissue sample; and

(iii) The razor cartridge is pressed downwardly forcing the cutting edges of the plurality of razor blades to press against and cut the tissue sample in the second orientation.

In at least one embodiment, the method of using the morcellating device further comprises rotating the plurality of razor blades to a third orientation relative to the tissue sample, and then pressing the razor cartridge down onto the circular sample tray holder, thereby forcing the cutting edges of the plurality of razor blades to press onto the tissue sample in the third orientation and cut the tissue sample; and optionally, the method further comprises rotating the plurality of razor blades to a fourth orientation relative to the tissue sample and then pressing the razor cartridge down on the circular sample tray holder, thereby forcing the cutting edges of the plurality of razor blades to press on and cut the tissue sample in the fourth orientation. Optionally, in the method, the plurality of razor blades rotates at least 10 to 25 degrees per revolution.

In at least one embodiment, the present disclosure provides a kit for preparing a biological sample, wherein the kit comprises: immobilization reagents, cell dissociation reagents and assay reagents. In at least one embodiment, the kit further comprises a unfixed agent. In at least one embodiment, the kit further comprises a shredding device. In at least one embodiment, the present disclosure provides a kit comprising a shredding device of the present disclosure, and one or more razor blades in one or more containers.

Drawings

A better understanding of the novel features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also referred to herein as "figures") of which:

fig. 1 shows an example of a microfluidic channel structure for separating individual biological particles.

Fig. 2 shows an example of a microfluidic channel structure for delivering barcode-bearing beads to droplets.

Fig. 3 shows an example of a microfluidic channel structure for co-separating biological particles and reagents.

Fig. 4 shows an example of a microfluidic channel structure for controlled separation of beads into discrete droplets.

Fig. 5 shows an example of a microfluidic channel structure for achieving increased droplet generation throughput.

Fig. 6 shows another example of a microfluidic channel structure for achieving increased droplet generation throughput.

Fig. 7 shows an exemplary bar code bearing bead.

Fig. 8 shows another exemplary bar code carrying bead.

Fig. 9 shows an exemplary microwell array schematic.

FIG. 10 shows an exemplary microwell array workflow for processing nucleic acid molecules.

Fig. 11 schematically shows an example of the marking agent.

Fig. 12 depicts an example of a bead carrying a bar code.

Fig. 13A, 13B and 13C schematically depict an example workflow for processing nucleic acid molecules.

Fig. 14A, 14B and 14C depict a comparison plot of the percentage of total genes detected and total UMI detected in a fixed sample relative to a fresh control sample prepared and analyzed as described in example 2. Fig. 14A depicts a graph of the results of "post-dissociation fixed" adult mouse kidney tissue on days 3 and 7 post-fixation; FIG. 14B depicts a graph of the results of "cut-fixed" adult mouse kidney tissue on days 1 and 6 after fixation; fig. 14C depicts a graph of the results of "post-dissociation fixed" adult mouse brain tissue on days 1 and 6 post-fixation.

FIGS. 15A and 15B depict a comparison of ATAC transposition events from a minced-fixed E18 brain tissue nucleus sample prepared as described in example 4.

FIG. 16 depicts a comparison of detected cell type clusters in kidney tissue samples that were minced-fixed, dissociated, and unfixed as described in example 5, and stored for up to 6 days.

FIG. 17 shows a transposase-nucleic acid complex comprising a transposase, a first double stranded oligonucleotide and a second double stranded oligonucleotide, wherein the first double stranded oligonucleotide comprises a transposon end sequence and a first primer sequence and the second double stranded oligonucleotide comprises a transposon end sequence and a second primer sequence.

FIG. 18 shows a transposase-nucleic acid complex comprising a transposase, a first double stranded oligonucleotide comprising a transposon end sequence and first and second primer sequences, and a second double stranded oligonucleotide comprising a transposon end sequence and third and fourth primer sequences.

FIG. 19 shows a transposase-nucleic acid complex comprising a transposase, a first hairpin molecule and a second hairpin molecule.

Fig. 20 shows a schematic workflow of tandem ATAC and RNA processing.

Fig. 21 depicts a perspective side view of an exploded view of one embodiment of a mechanical shredding device (including a sample tray) of the present disclosure.

FIG. 22 depicts a top view of the shredder system of FIG. 21 after assembly.

Fig. 23A depicts a side view of one embodiment of the mechanical shredding device of the present disclosure in a stationary or normal position. Fig. 23B is a side view of the shredder of fig. 23A in the compressed slicing position. Fig. 23C is a perspective side view of the shredder of fig. 23A in a rest or normal position. Fig. 23D is a perspective side view of the shredding device of fig. 23A in a compressed position.

Fig. 24A is a side view of a shredder apparatus in a rest or normal position according to another embodiment. Fig. 24B is a side cross-sectional view of the shredding device of fig. 24A in a compressed position.

Fig. 25 is a side cross-sectional view of a shredder apparatus in a rest or normal position ("rest mode") according to another embodiment.

FIG. 26 is a side view of an exploded unassembled cutting device system including a sample tray and razor blades according to another embodiment of the present invention.

Fig. 27A is a top view of an alignment layer component of a shredding device according to a preferred embodiment of the present invention. Fig. 27B is a side cross-sectional view taken along line A-A of fig. 27A. Fig. 27C is a side view of fig. 27A. Fig. 27D is a bottom view of fig. 27A.

Fig. 28A is a top perspective view of an alignment layer assembly according to another embodiment of the present invention. Fig. 28B is a bottom perspective view of the alignment layer assembly of fig. 28A.

Fig. 29A, 29B, 29C, 29D, 29E and 29F depict a comparison of clusters of cell types detected in human uterine tissue samples that were dissociated, some of which were first cut-fixed, and/or snap frozen, and/or stored for up to 5 days as described in example 7, and others of which were not first subjected to the procedure described in example 7.

Detailed Description

For the purposes of the description herein and the claims that follow, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes more than one protein, and reference to "a compound" refers to more than one compound. It should also be noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements or the use of "negative" limitations. The use of "including," "comprising," "having," and "including" are interchangeable and are not intended to be limiting. It will also be understood that where the description of various embodiments uses the term "comprising," those skilled in the art will understand that in some specific instances, embodiments may alternatively be described using a language that "consists essentially of or" consists of.

Where a range of values is provided, unless the context clearly dictates otherwise, it is understood that each intermediate integer of a value between the upper and lower limits of that range and each tenth of the value (unless the context clearly dictates otherwise) and any other statement or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the present disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of these limits, ranges excluding either (i) or (ii) of those included limits are also included in the disclosure. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", and the like.

Generally, the nomenclature used herein and the techniques and procedures described herein include those well understood and commonly employed by those of ordinary skill in the art, such as those commonly employed in, for example, the following documents: green and Sambrook the number of the individual pieces of the plastic,Molecular Cloning:A Laboratory Manual(fourth edition), volumes 1 to 3, cold Spring Harbor Laboratory, cold Spring Harbor, n.y.,2012 (hereinafter "Sambrook"); and Current Protocols in Molecular BiologyAusubel et al, edited, F.M., initially by Greene Publishing Associates, inc. and John Wiley, 1987&Sons, inc. Published in book form, and then regularly supplemented until 2011, is currently available online in journal format:current Protocols in Molecular Biology, the first Rolls 00 to 130(1987-2020), by Wiley&Sons, inc. Published under Wiley Online Library (hereinafter "Ausubel").

All publications, patents, patent applications, and other documents cited in this disclosure are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. For the purposes of explaining the present disclosure, the following description of terms will apply, and where appropriate, terms used in the singular will also include the plural and vice versa.

A. Overview of the shredding-fixing method

Herein is recognized a need for methods, compositions, kits, and systems for analyzing a variety of cellular analytes (e.g., genomic, epigenomic, transcriptomic, metabolomic, and/or proteomic information) present in biological samples obtained from tissues. The ability to accurately determine biological samples from tissue requires the rapid and efficient release of a wide variety of cell types from the tissue in order to be able to obtain relevant cellular analyte information (e.g., RNA transcripts) for each of the various cell types before degradation occurs. Ideally, the state of the cellular analyte released from the various cell types in the tissue is not significantly altered from its natural environment (i.e., the state of the cellular analyte in the cells within the tissue matrix prior to treatment to release the cellular analyte). Typical methods of releasing cellular analytes from biological tissue samples for assay involve the use of certain combinations of lysing agents, enzyme inhibitors, chelating agents, physical agitation and heating to facilitate the activity of the various reagents involved.

For example, practitioners of single cell assays for transcriptome analysis generally believe that exposure of a whole unfixed tissue sample to room temperature conditions (e.g., after removal from ice) results in rapid degradation of RNA transcripts present within the tissue. There are two main reasons for the rapid degradation of RNA transcripts during tissue sample preparation prior to analysis. First, RNA is composed of ribose units with highly reactive C2 hydroxyl groups, which makes RNA chemically less stable and more susceptible to thermal degradation than DNA. Second, enzymes that degrade RNA (e.g., ribonucleases) are ubiquitous and removal thereof prior to or during sample preparation is almost impossible. In addition, damaged and rotted eukaryotic tissues are known to produce more ribonucleases. Thus, human tissue that has undergone surgical removal and is allowed to stand at room temperature is very difficult to work with in a workflow for single cell assays using RNA transcripts.

The present disclosure provides methods of improving the release of cellular analytes from biological tissue samples that allow for analysis of various populations of different cell types present in the natural environment of such tissues. Biological samples prepared using these methods allow improved assays, including partition-based assays, to be performed using fixed cells or nuclei from such tissue samples, with little or no artifacts in the obtained cellular analyte information. Standard methods known in the art prepare samples for partition-based analysis by treating whole tissue samples with dissociating or fixing reagents to provide isolated cells for analysis. However, these methods produce samples that provide significantly different cellular analyte measurements than fresh samples. Notably, certain rare cell types found in freshly prepared cells from tissue samples were not detected. A surprising advantage of the present disclosure is that the methods of using minced and fixed tissue samples disclosed herein provide biological samples for zonal-based analysis that retain cellular analytes from rare cell types found in tissue (e.g., neuronal cell types from brain tissue). The shredding and fixation treatments disclosed herein can provide a cell or nucleus sample from a tissue for use in batch assays or single cell/single cell nucleus assays, such as partition-based assays of nucleic acid sequences present in the cells or nuclei.

The present disclosure provides a general method for preparing a biological sample comprising: (a) shredding biological tissue to form a tissue fragment composition; (b) Treating the tissue fragment composition with a fixation reagent, thereby providing a fixed tissue fragment composition; and (c) treating the fixed tissue fragment composition with a cell dissociation agent. This chopper-immobilized treatment, along with the cell dissociation reagents, provides an immobilized cell or nucleus composition from the tissue that includes a distribution of various cell types from the tissue, which can then be used for further bulk analysis or partition-based analysis of cellular analytes. Such methods for batch analysis or partition-based analysis of cells or nuclei (e.g., single cell/single cell nuclear analysis of DNA and/or RNA sequences) that can be used with the cut-to-fix method of preparing a biological sample are provided elsewhere herein.

The "biological tissue" used in the methods of the present disclosure may be any tissue of biological origin, including tissue from a tissue specimen, a biopsy, a solid tissue sample, or tissue from a tissue culture. The tissue sample may comprise: tissue samples that have been manipulated (such as by treatment with a reagent) after separation from a biological source, or that have been embedded in a medium (e.g., paraffin), sectioned tissue samples (e.g., sectioned samples mounted on a solid substrate such as a slide), washed tissue, and/or tissue enriched for certain cell populations (such as cancer cells, neurons, stem cells, etc.), tissue obtained by surgical excision, tissue obtained by biopsy, tissue samples from organs, bone marrow, blood, plasma, serum, etc. It is contemplated that the biological tissue used in the methods of the present disclosure may be derived from another sample. The biological tissue sample may comprise tissue obtained by core biopsy, needle aspiration or fine needle aspiration.

Biological tissue that may be used with the methods of the present disclosure include: brain tissue, skin tissue, muscle tissue, smooth muscle tissue, cardiac muscle tissue, skeletal muscle tissue, bone marrow tissue, lung tissue, bronchus tissue, fallopian tube tissue, gall bladder tissue, ovary tissue, testis tissue, hypothalamus tissue, thyroid tissue, adrenal tissue, kidney tissue, pancreas tissue, small intestine tissue, large intestine tissue, colon tissue, liver tissue, lymph tissue, breast tissue, mesenteric tissue, nose tissue, pineal tissue, parathyroid tissue, pharynx tissue, larynx tissue, pituitary tissue, prostate tissue, saliva tissue, spinal cord tissue, spleen tissue, stomach tissue, thymus tissue, trachea tissue, tongue tissue, urethra tissue, placenta tissue, artery tissue, vein tissue, and tonsil tissue. Furthermore, it is contemplated that biological tissue that may be used with the methods of the present disclosure may include cancers of any one or more of the foregoing biological tissue types.

A "biological sample" prepared using the method may include cells, biomolecules, such as nucleic acids, proteins, carbohydrates, lipids, and/or combinations of any of these. Biological samples may include cells and other biological material derived from tissue and/or cells, as well as cell-free samples. The cell-free sample may comprise extracellular polynucleotides.

The term "shredding," as used herein, refers to any mechanical activity that separates a piece of larger material (e.g., biological tissue) into a plurality of smaller pieces or fragments. Simply treating a whole tissue sample with a fixative and/or dissociation reagent does not result in a fixed cell or nucleus sample from the tissue, which provides results for cellular analytes representative of fresh tissue in a batch or zone-based assay. A range of mechanical methods and devices for morcellating tissue are available in the art and can be used in the methods of the present disclosure. Exemplary shredding methods and apparatus are provided elsewhere herein (including "embodiments").

The term "tissue fragments" refers to small pieces obtained from the cutting of tissue and is intended to include small pieces that are a mixture of cells and other biological materials that are characteristic of a certain tissue type. The tissue mass may include a plurality of cell types that are specific to a tissue type. While not intending to be bound by theory or a particular mechanism, it is believed that the shredding provides a tissue fragment composition that is capable of rapid fixation and subsequent dissociation without the presence of certain biological stresses that distort the distribution and/or performance of cell types and other cellular analytes from the tissue sample.

As disclosed elsewhere herein, the minced biological tissue provides a tissue fragment composition comprising particles having an average size on one side of about 500 μm or less, about 250 μm or less, about 125 μm or less, about 75 μm or less, or about 50 μm or less. Generally, a useful range of tissue fragment compositions comprises particles having an average size on one side of between about 50 μm and 500 μm, between about 125 μm and 500 μm, between about 250 μm and 500 μm, between about 50 μm and 250 μm, or between about 50 μm and 125 μm.

Further, as disclosed elsewhere herein, the shredding of biological tissue is performed on a smooth, flat surface at near freezing temperatures (e.g., between about 2 ℃ and 8 ℃) where the shredding action minimizes stress on the various cell types present in the tissue. For example, fresh brain tissue samples are placed in clean petri dishes maintained on ice, and the tissue is finely minced by hand with a single clean razor blade, with the step size between each cut kept as small as possible. The morcellation is performed with little or no dragging or rotating tissue. After sufficient chopping of the brain tissue sample on the petri dish surface in one complete dimension, the chopping operation is repeated in the same manner on at least 2 additional dimensional axes of the sample. This morcellation process produces tissue fragments of optimal size for rapid fixation and further dissociation with minimal thermally or mechanically induced stress that can lead to degradation of the cell contents. It is also contemplated that the shredding operation may be performed using a mechanical device that simulates the manual shredding process, including spacing and directionality along three different axes across the sample.

In at least one embodiment, the minced biological tissue operations used in the minced-fixing methods of the present disclosure can be performed using a mechanical "minced device" (or "tissue slicing device") as described elsewhere herein and in U.S. provisional patent application entitled "Tissue Slicer Devices And Methods Of Using The Same", U.S. serial No. 63/213,908, filing date 2021, 6 months 23, which is incorporated herein by reference in its entirety.

Tissue fragments produced by the chopping process may be immediately fixed by immersion in a fixative or fixative solution. "immobilized" refers to a state that is preserved against decay and/or degradation. While not intending to be bound by a particular theory or mechanism, in general, the immobilization process is a result of the biomolecules within the sample being in contact with the immobilization reagent for a time, thereby forming crosslinks between the biomolecules in the sample, thereby preventing degradation processes. The minced tissue is immersed in a fixative solution, typically overnight at about 4 ℃. "fixed tissue fragments" include the following fragments of tissue resulting from shredding: due to this treatment with the fixative reagent, these tissue fragments undergo fixation, at least to some extent, in their constituent cells and cellular analytes.

For example, an exemplary method of preparing fixed tissue fragments from minced brain tissue includes immediately immersing the minced tissue in a 1% to 4% fixation reagent Paraformaldehyde (PFA) solution, then storing in a sidedraw tube overnight at 4 ℃. Typically, the ratio of the volume of fixation reagent solution to the volume of tissue fragments is about 10:1. In some embodiments, the ratio of the volume of fixation reagent solution to the volume of tissue fragments may be at least about 2.5:1, at least about 5:1, at least about 7.5:1, at least about 15:1, or at least about 20:1. After overnight storage, the tube is centrifuged (e.g., at 500rcf for 5 minutes) and the solution supernatant is removed to yield fixed tissue fragments. These fixed tissue fragments can be stored for various amounts of time with minimal degradation prior to further dissociation and/or analysis (e.g., in a partition-based assay). In at least one embodiment, the fixed tissue fragments may be stored for at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 1 week, at least 1 month, at least 6 months, or more prior to use.

Typically, these fixed tissue fragments are resuspended with a cell resuspension buffer (e.g., 1% bsa+0.2u/mL ribonuclease inhibitor PBS solution) prior to cell dissociation. "cell dissociation" refers to the process of treating a larger solid composition comprising a plurality of cells and other biological material (e.g., fixed tissue fragments) to divide it into individual cells. In the context of the methods of the present disclosure, it is contemplated that cell dissociation of the fixed tissue fragments may be performed using any cell dissociation reagent solution known in the art. Because the tissue fragments include cells associated with a biological material comprising collagen, in at least one embodiment, the cell dissociation agent for treating the immobilized tissue fragments includes collagenase. For example, as disclosed elsewhere herein, the buffer in which the fixed brain tissue fragments are resuspended is removed and replaced with the dissociation agent collagenase a. The tube containing this cell dissociation reagent mixture of fixed tissue fragments is then shaken (e.g., at about 700rpm for about 90 minutes) at 37 ℃. In addition, dissociation of cells in the fixed tissue fragments may be facilitated by other physical means, such as repeated pipetting and centrifugation.

B. Use of cells or nuclei from the cut-fixation method in assays

The product of the cut-to-fix method used to prepare the biological sample is a preparation of dissociated fixed cells derived from the tissue sample or fixed nuclei derived from the tissue sample, which may be further processed and/or used in various analytical methods. The ability to use immobilized cells or nuclei in an assay requires that the preparation be performed quickly and efficiently so that the relevant cellular analytes are immobilized before degradation occurs. Ideally, the assay data obtained from the fixed cells or nuclei should resemble as closely as possible fresh samples obtained from their natural environment. The methods of the present disclosure for the chopper-immobilized preparation of tissue samples provide dissociated immobilized cells or immobilized nuclei from the tissue, which can be used in a range of assays.

Generally, each immobilized cell or nucleus produced by this method comprises a plurality of crosslinked nucleic acid molecules as a result of treatment with an immobilization agent. These crosslinks act to prevent degradation of nucleic acid molecules (particularly RNA transcripts) and thereby preserve cellular information associated with a particular population of nucleic acid molecules in a natural or fresh state. As described elsewhere herein, it is contemplated that an assay directed to detecting and/or measuring a population of nucleic acid sequences present in an immobilized cell or cell nucleus can be performed using an immobilized cell or cell nucleus composition from a tissue sample prepared by a chopper-immobilized method. For example, in at least one embodiment, the present disclosure provides a method of biological tissue analysis, wherein the method comprises: (a) Chopping biological tissue to provide a tissue fragment composition; (b) Treating the tissue fragment composition with a solution comprising a fixative to provide a fixed tissue fragment composition; (c) Treating the fixed tissue fragment composition with a cell dissociation agent to provide fixed cell compositions, each fixed cell or cell nucleus comprising a plurality of crosslinked nucleic acid molecules; and (d) generating a plurality of barcoded nucleic acid molecules from the plurality of crosslinked nucleic acid molecules and the plurality of nucleic acid barcode molecules, wherein one barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules comprises i) a sequence corresponding to one crosslinked nucleic acid molecule of the plurality of crosslinked nucleic acid molecules, or a complement thereof, and ii) a barcode sequence, or a complement thereof. This general method of generating barcoded nucleic acid molecules from crosslinked nucleic acids present in an immobilized cell or immobilized cell nucleus composition can be performed using a variety of partition-based assays described in more detail elsewhere herein (including "examples").

It is a feature of the methods of the present disclosure to be able to prepare dissociated immobilized cells or immobilized nuclei of biological samples for assays starting from tissue samples. "immobilized biological sample" refers to a biological sample that has been contacted with an immobilized reagent. For example, formaldehyde-immobilized biological samples have been contacted with the immobilization reagent formaldehyde. "fixed cells", "fixed nuclei" or "fixed tissue" refer to cells, nuclei or tissue that have been contacted with a fixative under conditions sufficient to permit or cause intramolecular and intermolecular covalent crosslinks to form between biomolecules in a biological sample.

The amount of time that the biological sample is contacted with the fixative to provide a fixed biological sample depends on the temperature, sample properties, and fixative used. For example, the biological sample may be contacted with the immobilized reagent for 72 hours or less (e.g., 48 hours or less, 24 hours or less, 18 hours or less, 12 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, 60 minutes or less, 45 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, or 2 minutes or less).

In general, contact of a biological sample (e.g., a tissue fragment) with an immobilizing reagent (e.g., paraformaldehyde or PFA) causes intramolecular and intermolecular covalent crosslinks to form between biomolecules in the biological sample. In some cases, the immobilization reagent formaldehyde is known to produce covalent aminal crosslinks within RNA, DNA, and/or protein molecules. Examples of fixative agents include, but are not limited to, aldehyde fixatives (e.g., formaldehyde, also commonly referred to as "paraformaldehyde," "PFA," and "formalin"; glutaraldehyde; etc.), imide esters, NHS (N-hydroxysuccinimide) esters, and the like.

The formation of crosslinks in biomolecules (e.g., proteins, RNA, DNA) due to immobilization can affect the ability to detect (e.g., bind, amplify, sequence, hybridize) the biomolecules in standard assay methods, methods for removing crosslinks or unfixing them are described elsewhere herein. However, some assays can detect and provide useful information from fixed cells or nuclei. The widely used fixative reagents paraformaldehyde or PFA fix tissue samples by catalyzing the formation of crosslinks between basic amino acids (such as lysine and glutamine) in proteins. Intramolecular and intermolecular crosslinks can be formed in the protein. These crosslinks preserve the secondary structure of the protein and also eliminate enzymatic activity in the preserved tissue sample.

The present disclosure provides methods and kits for preparing a biological sample of fixed cells or nuclei from biological tissue, which can be used in assays for detecting and/or measuring a range of cellular analytes. Suitable cellular analytes include, but are not limited to, intracellular/nuclear analytes and extracellular analytes. The cellular analyte may be a protein, metabolite, metabolic byproduct, antibody or antibody fragment, enzyme, antigen, carbohydrate, lipid, macromolecule, or combination thereof (e.g., proteoglycan), or another biological molecule. The cellular analyte may be a nucleic acid molecule. The cellular analyte may be a deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA) molecule. The DNA molecule may be a genomic DNA molecule. The cellular analyte may comprise coding or non-coding RNA. The RNA may be, for example, messenger RNA (mRNA), ribosomal RNA (rRNA), or transfer RNA (tRNA). The RNA may be a transcript. The RNA may be a small RNA less than 200 nucleobases in length, or a large RNA greater than 200 nucleobases in length. The micrornas can include 5.8S ribosomal RNAs (rrnas), 5S rrnas, transfer RNAs (trnas), micrornas (mirnas), small interfering RNAs (sirnas), small nucleolar RNAs (snornas), RNAs that interact with Piwi proteins (pirnas), tRNA-derived micrornas (tsrnas), and small rDNA-derived RNAs (srrnas). The RNA may be double-stranded RNA or single-stranded RNA. The RNA may be circular RNA.

In some cases, the cellular analyte associates with an intermediate entity, wherein the intermediate entity is analyzed to provide information about the cellular analyte and/or the intermediate entity itself. For example, an intermediate entity (e.g., an antibody) may bind to an extracellular analyte (e.g., a cell surface receptor) or a nuclear membrane protein, wherein the intermediate entity is treated to provide information about the intermediate entity, the extracellular analyte, or both. In one embodiment, the intermediate entity includes an identifier (e.g., a barcode molecule) that can be used to generate a barcode molecule (e.g., a barcode based on a droplet), as further described herein.

In some embodiments, the immobilized cells or nuclei resulting from the chopper-immobilization method are immobilized by treatment with formaldehyde. The term "formaldehyde" when used in the context of fixatives also refers to "paraformaldehyde" (or "PFA") and "formalin," both of which have particular meanings in connection with formaldehyde compositions (e.g., formalin is a mixture of formaldehyde and methanol). Thus, formaldehyde-fixed tissue fragments or cell/nuclei may also be referred to as formalin-fixed or PFA-fixed. Protocols and methods for preparing immobilized biological samples using formaldehyde as an immobilization reagent are well known in the art and can be used in the methods of the present disclosure. For example, suitable ranges for formaldehyde concentrations for preparing a fixed biological sample are 0.1% to 10%, 1% to 8%, 1% to 4%, 1% to 2%, 3% to 5%, or 3.5% to 4.5%. In at least one embodiment of the shredder-fixation method of the present disclosure, formaldehyde is used to fix tissue fragments at a final concentration of 1%, 4% or 10%. Typically, formaldehyde is diluted from a more concentrated stock solution (e.g., 35%, 25%, 15%, 10%, 5% PFA stock solution).

In at least one embodiment, the method of preparing a biological sample using a chopper-fixation treatment of biological tissue uses PFA as a fixative, and stabilization of the fixation allows dissociation of the fixed cells or nuclei from the tissue after fixation, and/or further determination of the amount of time before fixation of the cells or nuclei is at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 1 week, at least 1 month, at least 6 months, or more.

C. De-pinning process

In some assay methods, it may be useful to use cells or nuclei that do not include cross-linking caused by immobilization. In such methods, it may be preferable to prepare the unfixed cells or nuclei. "unfixed" refers to a state of processing of a cell, nucleus, plurality of cells, plurality of nuclei, tissue sample, or any other biological sample, characterized by a previously fixed state followed by a reversal of the previously fixed state. The unfixed cells or nuclei are characterized by cleavage or reversal of covalent bonds in the cells/nuclei or biomolecules of the sample, wherein such covalent bonds were previously formed by treatment with an immobilizing agent (e.g., paraformaldehyde or PFA). Unfixed cells or unfixed nuclei may also be referred to as "previously fixed" cells or nuclei.

The present disclosure contemplates that dissociated fixed cells or nuclei obtained from the minced-fixed tissue sample may be further processed to provide dissociated fixed cells or nuclei. Thus, in at least one embodiment, the shredder-immobilization method may further comprise treating the dissociated, immobilized cells or nuclei from the tissue sample with a solution comprising a deammobilizing agent.

The term "unfixed agent" (or "de-crosslinking agent") as used herein refers to a compound or composition that reverses the immobilization and/or removes crosslinks within or between biomolecules in a sample resulting from prior use of the fixative. In some embodiments, the unfixed agent is a compound that catalyzes the removal of crosslinks in the fixed sample. Exemplary compounds (1) to (15) useful as a unfixed agent in the methods of the present disclosure include the compounds of table 1 below. Furthermore, methods of preparing and using a disaggregating agent, such as compounds (1) to (15), to prepare biological samples from immobilized cells for batch and partition-based assays of cellular analytes are disclosed in International patent application No. PCT/US2020/066701 filed on month 22 of 2020, which is hereby incorporated by reference.

Table 1: exemplary Defixative Compounds

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At least one of the disaggregating agents in Table 1, namely compound (3), has been previously shown to be capable of catalyzing the decomposition of aminal and hemi-aminal adducts formed in formaldehyde-treated RNA and is compatible with many RNA extraction and detection conditions. See, e.g., karmakar et al, "Organocatalytic removal of formaldehyde adducts from RNA and DNA bases," Nature Chemistry,7:752-758 (2015); and US2017/0283860A1.

Proline is a unique amino acid that contains a secondary amine in the 5-membered ring, resulting in high nucleophilicity. The high nucleophilicity in the proline analog structures of compounds (12), (13), (14) and (15), together with the adjacent amine or acid moieties, suggests that these compounds, such as compounds (1) to (11), may also be used to catalyze the decomposition of aminal and hemi-aminal adducts formed in formaldehyde-fixed RNAs and other biomolecules.

Compounds (1) to (6), (12) and (14) are commercially available. Compounds (7), (8), (9), (10), (11), (13) and (15) can be prepared from commercially available reagents using standard chemical synthesis techniques well known in the art. See, e.g., crisalli et al, "Importance of ortho Proton Donors in Catalysis of Hydrazone Formation," org. Lett.2013,15,7,1646-1649.

Compound (8) can be prepared by two steps as described in example 4. Briefly, in the preparation of compound (8), diethyl (4-aminopyridin-3-yl) phosphonate was prepared according to the procedure described in Guilar, R.et al Synthesis,2008,10,1575-1579. The target compound (8), i.e., (4-aminopyridin-3-yl) phosphonic acid, is then prepared by acid hydrolysis of the precursor compound diethyl (4-aminopyridin-3-yl) phosphonate. Similarly, compounds (9) and (10) can be prepared by a simple procedure. For example, compound (9) can be prepared in two steps as shown in the scheme below from 2-bromopyridin-3-amine (CAS registry number 39856-58-1; sigma-Aldrich, st. Louis, MO).

Compound (10) was similarly prepared from 4-bromopyrimidin-5-amine (CAS accession number 849353-34-0; ambeed, inc., arlington Heights, IL, USA) as shown in the scheme below in two steps.

Proline analog compounds (13) and (15) were prepared from commercially available protected precursor compounds by direct single-step deprotection.

Thus, in embodiments of the present disclosure that include a step of unfixing a fixed cell or nucleus, the unfixed agent used may include a compound selected from table 1. For example, the unfixed agent may include any of the following compounds: compound (1), compound (2), compound (3), compound (4), compound (5), compound (6), compound (7), compound (8), compound (9), compound (10), compound (11), compound (12), compound (13), compound (14), compound (15), or a combination of one or more of the compounds of table 1.

In at least one embodiment of the method, the tissue fragments are immobilized with PFA, and the deagglomerating agent used in the solution is capable of removing crosslinks formed in the biological molecule by immobilization with PFA. In at least one embodiment, the unfixed agent comprises a compound selected from the group consisting of: compound (1), compound (2), compound (3), compound (4), compound (5), compound (6), compound (7), compound (8), compound (9), compound (10), compound (11), compound (12), compound (13), compound (14), compound (15), or a combination thereof; optionally, wherein the detackifier comprises a compound selected from the group consisting of: compound (1), compound (8), or a combination thereof.

In at least one embodiment, treating the fixed cell or cell nucleus composition with a disarming agent may further comprise incubating the fixed cell or cell nucleus with a protease, optionally a cold active protease. In at least one embodiment, the dissociating immobilized cells or nuclei produced by the chopper-immobilized method are subjected to a dissociation immobilization treatment comprising incubating the cells or nuclei with a combination of a dissociation agent and a protease. Methods for preparing biological samples from immobilized cells for batch and partition-based assays of cellular analytes using cold active proteases, optionally together with a disaggregating agent, are disclosed in international patent application No. PCT/US2021/026592 filed on 9/4/2021, which is hereby incorporated by reference.

A wide variety of proteases are known in the art that can be used as lysing agents, and that can be used to release cellular analytes from cells or nuclei, tissue samples, and other types of biological samples. Although these proteases are used in methods that are practiced above room temperature, typically at temperatures above 37 ℃, it is contemplated that cold active (or psychrophilic) proteases may be used in the context of the methods of the present disclosure. Cold active proteases exhibit at least some measurable proteolytic activity at temperatures as low as 0 ℃ and typically exhibit significant proteolytic activity in the range between about 5 ℃ and about 15 ℃. As described above, even proteases exhibiting peak activity at much higher temperatures, such as subtilisin a, may have sufficiently high low temperature activity to be used as cold active proteases in the methods of the present disclosure. In at least one embodiment of the method of the invention, the protease has the following average activity at a temperature between about 5 ℃ and about 15 ℃): at least 1.0U/mg, at least 5.0U/mg, 10.0U/mg, at least 50U/mg, at least 100U/mg, or higher. The average protease activity at a temperature between about 5 ℃ and about 15 ℃ can be determined by the ordinarily skilled artisan using, for example, well-known universal protease activity assays employing casein substrates and foggy-tin two's reagent. Reagents and kits for performing such protease activity assays are commercially available (e.g., from Millipore-Sigma; USA).

Thus, in at least one embodiment of the method, the protease used in the method is a cold active protease; optionally, wherein the protease has an average activity of at least 1.0 units/mg protease at a temperature between about 5 ℃ and about 15 ℃. In some embodiments, the protease has maximum activity at a temperature between about 50 ℃ and about 60 ℃. Furthermore, in some embodiments of the method, the temperature and time of incubation are expected to vary somewhat based on the particular protease used, and such conditions may be optimized by the ordinarily skilled artisan.

It is also contemplated that the amount of protease used in the treatment may be varied in order to adjust the low temperature proteolytic activity to an effective level. Thus, in at least one embodiment of the method, the protease concentration in the solution is between about 1mg/mL and 100 mg/mL; optionally, the protease concentration in the solution is between about 5mg/mL and 10 mg/mL.

In at least one embodiment of the method, the protease is a serine protease (e.c. 3.4.21); optionally, wherein the serine protease is selected from chymotrypsin-like, tryptase-like, thrombin-like, elastase-like and subtilisin-like A protease. A wide variety of different serine proteases have been well characterized and are commercially available. Serine proteases that can be used in the methods of the present disclosure are selected from: alcalase, alkaline proteases, arcticzes proteases (ArcticZymes Technologies ASA,norway), bacitracin A, bacitracin B, bioprase, colistinase, esperase, genenase, kazusase, maxatase, proteinase K, proteinase S, savinase, serratia (Serratia) peptidase (i.e., a peptidase derived from an unknown species of Serratia sp.), subtilisin A, subtilisin B, subtilisin BL, subtilisin E, subtilisin J, subtilisin S41, thermostable protease, and trypsin.

Proteases have different substrate preferences, and thus mixtures of proteases are often used to release cellular analytes or other biological materials from cells or nuclei. Thus, in some embodiments, it is contemplated that the low temperature protease treatment may comprise incubating the immobilized biological sample with a protease composition. In at least one embodiment, the methods of the present disclosure may be implemented as: wherein the biological sample is incubated with a cryogenically active protease composition comprising at least two different proteases. In some embodiments, the composition comprises at least two proteases selected from the group consisting of: alcalase, alkaline protease, arcticzes protease, bacitracin a, bacitracin B, bioprase, colistinase, esperase, genenase, kazusase, maxatase, proteinase K, proteinase S, savinase, serratia peptidase (i.e., a peptidase derived from an unknown species of serratia), subtilisin a, subtilisin B, subtilisin BL, subtilisin E, subtilisin J, subtilisin S41, thermostable protease, and trypsin. For example, in at least one embodiment, a cryogenically active protease composition useful in the methods of the present disclosure comprises subtilisin a and proteinase K.

The process of preparing a disaggregated cell or nucleus sample from a previously immobilized biological sample using a disaggregating agent and a low temperature protease typically involves incubating the sample in an aqueous solution containing the protease at a temperature between about 5 ℃ and about 15 ℃ for at least one hour. In another embodiment, the incubation is for a period of time between 1 hour and 3 hours. In some embodiments, the solution further comprises a de-fixative that reverses cross-linking between biomolecules of the sample during the low temperature incubation period. It is also contemplated that in some embodiments, the application of short periods of heat and physical agitation to the sample after incubation may aid in the sample preparation process without creating artifacts associated with standard high temperature protease treatment. Thus, in at least one embodiment, the method of the present disclosure may be implemented as follows: wherein, after incubating the immobilized cells or the nuclear composition with the unfixed agent and the protease, the solution is shaken at a temperature between about 65 ℃ and 75 ℃ for at least 15 minutes.

D. Use in batch and partition-based assay methods

The disclosed methods using a shredder-fixation treatment of a tissue sample may be used to prepare dissociated fixed or unfixed cell samples from the tissue for use in a range of assay methods. Such assay methods may include "batch" assays using relatively large sample volumes, or single cell/single cell nuclear assays, such as zone-based (or droplet-based) assays. Exemplary single cell RNA profiling assays that can be used with the sample prepared by the chopper-fixation of the present disclosure are described in the "examples," including the fixed RNA profiling assays described in patent publications WO2021041974A1, WO2019165318A1, and US20200239874A1, each of which is hereby incorporated by reference in its entirety.

The universal cut-to-fix method for preparing biological samples from tissue as described above and elsewhere herein provides a sample of dissociated fixed cells from the tissue that can be further disarmed or used as such in a variety of batch assays. Accordingly, in at least one embodiment, the present disclosure also provides a method of bulk analysis of biological tissue, wherein the method comprises: (a) Chopping biological tissue to provide a tissue fragment composition; (b) Treating the tissue fragment composition with a solution comprising a fixative to provide a fixed tissue fragment composition; (c) Treating the fixed tissue fragment composition with a cell dissociation agent to provide a fixed cell or cell nucleus composition; and (d) further using the immobilized cell or cell nucleus composition as an input sample for cellular analyte assays. As described above, the immobilized cells or nuclear composition optionally may be treated with a destabilizing agent prior to further use in the assay. It is contemplated that any of a wide range of batch assays that are well known in the art and that can use dissociated cells as input samples can be used in this context to detect and quantify cellular analytes.

The use of dissociated fixed cells or nuclei derived from a tissue sample in a zone-based assay creates additional challenges because of the small sample size and the need to perform the assay with very small sample volumes while maintaining physical separation of the samples. As used herein, the term "partition" generally refers to a space or volume that may be suitable for containing one or more species or carrying out one or more reactions. The partitions may be physical compartments, such as droplets or wells (e.g., microwells). A partition may isolate a space or volume from another space or volume. A partition may be a droplet of a first phase (e.g., an aqueous phase) in a second phase (e.g., oil) that is immiscible therewith. The partition may be a droplet of the first phase in a second phase that is not phase separated therefrom, such as a capsule or liposome in an aqueous phase. A partition may include one or more other (internal) partitions. In some cases, a partition may be a virtual compartment, which may be defined and identified by an index (e.g., an index library) that spans multiple and/or remote physical compartments. For example, the physical compartment may include a plurality of virtual compartments.

Preparing partitions of biological samples containing one or more fixed cells or nuclei that can be used in partition-based assays involves many steps (e.g., sample transport, tissue dissociation, liquid phase washing and transfer, library preparation) that typically take hours to days. During this preparation time, the unfixed biological sample will begin to degrade and break down, resulting in a significant loss of cellular analyte information, thereby producing a measurement that does not reflect the natural state of the sample.

One type of zone-based assay is a droplet-based assay. Biological samples used in such assays are separated and partitioned into discrete droplets in an emulsion. Discrete droplets typically include a sample unique identifier in the form of a unique oligonucleotide sequence also contained in the droplet. The discrete droplets may further comprise an assay reagent for generating a detectable analyte (e.g., a 3' cdna sequence) from the sample and providing useful information about the analyte (e.g., RNA transcript profile).

The methods of the present disclosure can be used to prepare a biological sample derived from fixed cells or fixed nuclei of tissue, encapsulated in discrete droplets along with a cryogenically active protease and a disarming agent. The combination of protease and a disarming agent in the droplet with the immobilized sample is capable of reversing the immobilized state of the biomolecules in the sample while the sample is sequestered in the droplet. Accordingly, in some embodiments, the present disclosure provides a method of preparing a biological sample comprising: discrete droplets are generated that encapsulate the immobilized biological sample, the protease composition, and the disaggregating agent. The method may further comprise the step of immobilizing the biological sample prior to generating the discrete droplets.

In at least one embodiment, the method further comprises generating discrete droplets of the encapsulated biological sample. In at least one embodiment, the method further comprises generating discrete droplets encapsulating the immobilized biological sample and the protease. In at least one embodiment, the method further comprises generating discrete droplets of the encapsulated immobilized biological sample, protease, and the de-immobilization agent.

In at least one embodiment wherein the method comprises generating discrete droplets, the discrete droplets further comprise an assay reagent; optionally, wherein the assay reagents are contained in a bead. In at least one embodiment, the discrete droplets further comprise a bar code; optionally, wherein the barcode is contained in a bead.

Methods, techniques, and protocols that can be used to separate biological samples (e.g., individual cells, individual nuclei, biomolecular content of cells, etc.) into discrete droplets are known in the art and are well described in the art. The discrete droplets produced act as containers on the order of nanoliters that can keep the droplet contents separate from the contents of the other droplets in the emulsion. Methods and systems for producing stable discrete droplets of individual particles from a biological sample encapsulated in a non-aqueous emulsion or an oil emulsion are described, for example, in U.S. patent application publication nos. 2010/0105112 and 2019/0100632, each of which is incorporated herein by reference in its entirety for all purposes. Briefly, discrete droplets encapsulating a biological sample are achieved in an emulsion by introducing a flow stream of an aqueous fluid containing the biological sample into a flow stream of a non-aqueous fluid immiscible therewith such that droplets are generated at the junction of the two streams (see fig. 1-3). By providing an aqueous stream at a concentration and/or flow rate of a biological sample, the occupancy of the resulting droplets can be controlled. For example, the relative flow rates of the immiscible fluids may be selected such that, on average, each discrete droplet contains less than one biological particle. Such a flow rate ensures that the occupied droplets are occupied primarily by a single sample (e.g., a single cell or a single nucleus). Discrete droplets of encapsulated biological sample are also achieved in emulsions using a microfluidic architecture comprising channel segments with channel connections to reservoirs (see fig. 4-6).

As used herein, the term "biological particle" generally refers to a discrete biological system derived from a biological sample. The biological particles may be macromolecules. The biological particles may be small molecules. The biological particle may be a virus. The biological particles may be cells or derivatives of cells. The biological particles may be organelles from cells. Examples of organelles from cells include, but are not limited to, nuclei, endoplasmic reticulum, ribosomes, golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytosis vesicles, vacuoles, and lysosomes. The biological particles may be rare cells from a population of cells. The biological particles can be any type of cell including, but not limited to, prokaryotic cells, eukaryotic cells, bacteria, fungi, plants, mammalian or other animal cell types, mycoplasma, normal tissue cells, tumor cells, or any other cell type whether derived from a single-cell organism or a multicellular organism. The biological particles may be a component of a cell. The biological particles may be or may include DNA, RNA, organelles, proteins, or any combination thereof. The biological particles may be obtained from a tissue of a subject. The biological particles may be hardened cells. Such hardened cells may or may not include cell walls or cell membranes. The biological particles may include one or more components of the cell, but may not include other components of the cell. Examples of such components are nuclei or organelles.

In some cases, a droplet of the plurality of discrete droplets formed in this manner comprises at most one particle (e.g., one bead, one cell, or one nucleus). The flow and microfluidic channel architecture can also be controlled to ensure that a single occupied droplet has a given number, unoccupied droplets are less than a certain level, and/or multiple occupied droplets are less than a certain level.

In another aspect of the disclosure, the immobilized cells or nuclei, the protease composition, and optionally the unfixed agent composition, can then be separated (e.g., in a droplet or well) from other reagents for processing one or more analytes as described herein. In one embodiment, the immobilized cells or nuclei, the protease composition, and optionally the unfixed agent composition, may be separated by a support (e.g., a bead) comprising a nucleic acid molecule suitable for barcoding one or more analytes. In another embodiment, the nucleic acid molecule may include a nucleic acid sequence that provides identification information, such as a barcode sequence.

As used herein, the term "barcode" generally refers to a label or identifier that conveys or is capable of conveying information about an analyte. The barcode may be part of the analyte. The barcode may be independent of the analyte. The barcode may be a tag attached to an analyte (e.g., a nucleic acid molecule) or a combination of the tag plus an inherent property of the analyte (e.g., the size of the analyte or terminal sequence). Bar codes may be unique. Bar codes can take a variety of different forms. For example, the barcode may comprise a polynucleotide barcode; random nucleic acid and/or amino acid sequences; and synthetic nucleic acid and/or amino acid sequences. The barcode can be attached to the analyte in a reversible or irreversible manner. The barcode may be added to a fragment of, for example, a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before, during, and/or after sequencing of the sample. Bar codes may allow for identification and/or quantification of individual sequencing reads.

As used herein, the term "barcoded nucleic acid molecule" generally refers to a nucleic acid molecule resulting from, for example, treatment of a nucleic acid barcode molecule with a nucleic acid sequence (e.g., a nucleic acid sequence complementary to a nucleic acid primer sequence comprised by the nucleic acid barcode molecule). The nucleic acid sequence may be a targeting sequence (e.g., targeted by a primer sequence) or a non-targeting sequence. For example, in the methods, compositions, kits, and systems described herein, nucleic acid molecules (e.g., messenger RNA (mRNA) molecules) of a cell or cell nucleus are hybridized and reverse transcribed with a nucleic acid barcode molecule (e.g., a nucleic acid barcode molecule containing a barcode sequence and a nucleic acid primer sequence complementary to the nucleic acid sequence of the mRNA molecule) to produce a barcoded nucleic acid molecule having a sequence corresponding to the nucleic acid sequence of the mRNA and the barcode sequence (or the reverse complement thereof). The barcoded nucleic acid molecules can serve as templates, such as template polynucleotides, which can be further processed (e.g., amplified) and sequenced to obtain target nucleic acid sequences. For example, in the methods and systems described herein, the barcoded nucleic acid molecules can be further processed (e.g., amplified) and sequenced to obtain the nucleic acid sequence of the mRNA.

As used herein, the term "bead" generally refers to a particle. The beads may be solid or semi-solid particles. The beads may be gel beads. The gel beads may include a polymer matrix (e.g., a matrix formed by polymerization or cross-linking). The polymer matrix may include one or more polymers (e.g., polymers having different functional groups or repeating units). The polymers in the polymer matrix may be randomly arranged, for example in a random copolymer, and/or have an ordered structure, for example in a block copolymer. Crosslinking may be achieved via covalent, ionic or induced interactions or physical entanglement. The beads may be macromolecules. Beads may be formed from nucleic acid molecules that are bound together. Beads may be formed via covalent or non-covalent assembly of molecules (e.g., macromolecules) such as monomers or polymers. Such polymers or monomers may be natural or synthetic. Such polymers or monomers may be or include, for example, nucleic acid molecules (e.g., DNA or RNA). The beads may be formed of a polymeric material. The beads may be magnetic or non-magnetic. The beads may be rigid. The beads may be flexible and/or compressible. The beads may be destructible or dissolvable. The beads may be solid particles (e.g., metal-based particles including, but not limited to, iron oxide, gold, or silver) covered with a coating comprising one or more polymers. Such coatings may be destructible or dissolvable.

Fig. 1 illustrates an exemplary microfluidic channel structure 100 that can be used to generate discrete droplets of particles (such as single cells or single nuclei) encapsulating a biological sample. The channel structure 100 may include channel segments 102, 104, 106, and 108 that communicate at a channel connection 110. In operation, a first aqueous fluid 112 comprising suspended particles (e.g., cells or nuclei) from a biological sample 114 is transported into the junction 110 along the channel segment 102, while a second fluid 116 (or "spacer fluid") that is immiscible with the aqueous fluid 112 is delivered from each of the channel segments 104 and 106 to the junction 110 to produce discrete droplets 118, 120 of the first aqueous fluid 112 that flow into the channel segment 108 and away from the junction 110. The channel segment 108 may be fluidly coupled to an outlet reservoir in which discrete droplets may be stored and/or harvested. The discrete droplets generated may include a single particle 114 (such as droplet 118) from the biological sample, or may generate discrete droplets (not shown in fig. 1) containing more than one particle 114. Discrete droplets may be free of biological particles 114 (such as droplets 120). Each discrete droplet is capable of maintaining its own content (e.g., a single biological particle 114) separate from the content of the other droplets.

Typically, the second fluid 116 comprises an oil, such as a fluorinated oil, that includes a fluorosurfactant that helps stabilize the resulting droplets. Examples of useful spacer fluids and fluorosurfactants are described, for example, in U.S. patent application publication No. 2010/0105112, which is incorporated herein by reference in its entirety for all purposes.

The microfluidic channels for generating discrete droplets illustrated in fig. 1 may be coupled to any of a variety of different fluid sources or receiving components, including reservoirs, tubes, manifolds, or other system fluid components. Furthermore, the microfluidic channel structure 100 may have other geometries, including geometries with more than one channel connection. For example, a microfluidic channel structure may have 2, 3, 4 or 5 channel segments each carrying biological particles, assay reagents and/or beads from a biological sample, which meet at a channel junction.

Generally, fluid for generating discrete droplets is directed to flow along one or more channels or reservoirs via one or more fluid flow units. The fluid flow unit may include a compressor (e.g., providing positive pressure), a pump (e.g., providing negative pressure), an actuator, etc., to control the flow of fluid. The fluid may also or alternatively be controlled via an applied pressure differential, centrifugal force, electric pumping, vacuum, capillary or gravity flow, or the like.

Those of ordinary skill in the art will recognize that many different microfluidic channel designs are available that can be used with the methods of the present disclosure to provide discrete droplets comprising biological particles from immobilized biological samples, protease compositions, unfixed agent compositions, and/or beads with bar codes and/or other assay reagents.

The inclusion of the bar code in the discrete droplets along with the biological sample provides a unique identifier that allows the data from the biological sample to be distinguished and analyzed separately. The bar code may be delivered before, after, or simultaneously with the biological sample in the discrete droplets. For example, the bar code may be injected into the droplet before, after, or simultaneously with the droplet generation. Barcodes useful in the methods of the present disclosure typically comprise nucleic acid molecules (e.g., oligonucleotides). Nucleic acid barcode molecules are typically delivered to the partition via a support (such as a bead). In some cases, the barcode nucleic acid molecules are initially associated with the beads when discrete droplets are generated and then released from the beads when a stimulus is applied to the droplets. The bar code bearing beads useful in the methods of the present disclosure are described in further detail elsewhere herein.

Methods and systems for dividing bar code bearing beads into droplets are provided in U.S. patent nos. 10480029, 10858702 and 10725027, U.S. patent publication nos. 2019/0367997 and 2019/0064173, and international application nos. PCT/US20/17785 and PCT/US20/020486, each of which is incorporated herein by reference in its entirety for all purposes.

Fig. 7 shows an example of a bead carrying a bar code. Nucleic acid molecules 702 (such as oligonucleotides) can be coupled to beads 704 by releasable linkages 706 (such as disulfide linkers). The same bead 704 may be coupled (e.g., via a releasable bond) to one or more other nucleic acid molecules 718, 720. Nucleic acid molecule 702 can be or comprise a barcode. As described elsewhere herein, the structure of a bar code may comprise a plurality of sequential elements. Nucleic acid molecule 702 can comprise functional sequence 708 that can be used in subsequent processing. For example, the functional sequence 708 may include a sequencer-specific flow cell junction sequence (e.g., forP5 sequence of the sequencing system) and sequencing primer sequences (e.g.for +.>R1 primer of a sequencing system). The nucleic acid molecule 702 can comprise a barcode sequence 710 for barcode a sample (e.g., DNA, RNA, protein, antibody, etc.). In some cases, the barcode sequence 710 may be bead-specific such that the barcode sequence 710 is common to all nucleic acid molecules (e.g., including the nucleic acid molecule 702) coupled to the same bead 704. Alternatively or in addition, the barcode sequence 710 may be Partition-specific such that barcode sequence 710 is common to all nucleic acid molecules coupled to one or more beads that are partitioned into the same partition. The nucleic acid molecule 702 can comprise a specific primer sequence 712, such as an mRNA-specific primer sequence (e.g., a poly-T sequence), a targeting primer sequence, and/or a random primer sequence. Nucleic acid molecule 702 can include an anchor sequence 714 to ensure that specific primer sequences 712 hybridize at the sequence ends (e.g., of mRNA). For example, the anchor sequence 714 can comprise a random short sequence of nucleotides, such as a 1-mer, 2-mer, 3-mer, or longer sequence, which can ensure that the poly-T fragment is more likely to hybridize at the sequence end of the poly-a tail of the mRNA.

Nucleic acid molecule 702 can comprise a unique molecular identification sequence 716 (e.g., a Unique Molecular Identifier (UMI)). In some cases, the unique molecular identification sequence 716 may comprise about 5 to about 8 nucleotides. Alternatively, the unique molecular identification sequence 716 may be compressed by less than about 5 or more than about 8 nucleotides. Unique molecule identification sequence 716 can be a unique sequence that varies between individual nucleic acid molecules (e.g., 702, 718, 720, etc.) coupled to a single bead (e.g., bead 704). In some cases, the unique molecular identification sequence 716 may be a random sequence (e.g., such as a random N-mer sequence). For example, UMI may provide a unique identifier of the captured starting mRNA molecule in order to allow quantification of the amount of RNA initially expressed. It should be appreciated that although fig. 7 shows three nucleic acid molecules 702, 718, 720 coupled to the surface of bead 704, individual beads may be coupled to any number of individual nucleic acid molecules, e.g., from one to tens to hundreds of thousands or even millions of individual nucleic acid molecules. The respective barcodes of individual nucleic acid molecules may comprise a common sequence segment or a relatively common sequence segment (e.g., 708, 710, 712, etc.) and a variable or unique sequence segment (e.g., 716) between different individual nucleic acid molecules coupled to the same bead.

Biological particles (e.g., cells or nuclei, immobilized cells or nuclei, de-immobilized cells or nuclei, DNA, RNA, etc.) can be co-partitioned along with the barcoded beads 704. The barcoded nucleic acid molecules 702, 718, 720 may be released from the beads 704 in the partitions. For example, in the context of analyzing sample RNA, the poly-T fragment (e.g., 712) of one of the released nucleic acid molecules (e.g., 702) can hybridize to the poly-a tail of an mRNA molecule. Reverse transcription can produce a cDNA transcript of mRNA, but the transcript includes each of the sequence segments 708, 710, 716 of the nucleic acid molecule 702. Since nucleic acid molecule 702 comprises anchor sequence 714, it is more likely to hybridize to the sequence end of the poly-A tail of mRNA and initiate reverse transcription. Within any given partition, all cDNA transcripts of individual mRNA molecules may contain one common barcode sequence fragment 710.

However, transcripts made from different mRNA molecules within a given partition may vary at unique molecular identification sequence 712 fragments (e.g., UMI fragments). Advantageously, even after any subsequent amplification of the contents of a given partition, the amount of different UMIs may be indicative of the amount of mRNA originating from the given partition, and thus the amount of mRNA originating from biological particles (e.g., cells or nuclei, fixed cells or nuclei, unfixed cells or nuclei, etc.). As described above, transcripts can be amplified, purified and sequenced to identify the sequence of cDNA transcripts of mRNA, as well as to sequence barcode and UMI fragments. Although a poly-T primer sequence is described, other targeting or random primer sequences may be used to initiate a reverse transcription reaction. Also, while described as releasing barcoded oligonucleotides into a partition, in some cases, nucleic acid molecules that bind to beads (e.g., gel beads) can be used to hybridize and capture mRNA on a bead solid phase, e.g., to facilitate separation of RNA from other cellular content. In such cases, further processing may be performed in the partition or outside the partition (e.g., in bulk). For example, the RNA molecules on the beads may undergo reverse transcription or other nucleic acid processing, additional adaptor sequences may be added to the barcoded nucleic acid molecules, or other nucleic acid reactions (e.g., amplification, nucleic acid extension) may be performed. The beads or products thereof (e.g., barcoded nucleic acid molecules) may be collected from the partitions and/or pooled together prior to being subjected to cleaning and further characterization (e.g., sequencing). The operations described herein may be performed in any useful or convenient step. For example, beads comprising nucleic acid barcode molecules may be introduced into a partition (e.g., a well or droplet) before, during, or after introduction of a sample into the partition. The nucleic acid molecules of the sample may be barcoded, which may occur on the beads (in case the nucleic acid molecules remain coupled to the beads) or after release of the nucleic acid barcode molecules into the partitions. Where nucleic acid molecules from the sample remain attached to the beads, the beads from the various partitions may be collected, pooled, and then subjected to further processing (e.g., reverse transcription, attachment of adaptors, amplification, clearance, sequencing). In other cases, the processing may occur in a partition. For example, conditions sufficient to perform barcoding, adaptor ligation, reverse transcription or other nucleic acid processing operations may be provided in the partitions, and these operations performed prior to cleaning and sequencing.

Fig. 8 shows another example of a bead carrying a bar code. Nucleic acid molecules 805 (such as oligonucleotides) can be coupled to the beads 804 via releasable linkages 806 (such as disulfide linkers). The nucleic acid molecule 805 may comprise a first capture sequence 860. The same bead 804 may be coupled (e.g., via a releasable bond) to one or more other nucleic acid molecules 803, 807 comprising other capture sequences. The nucleic acid molecule 805 may be or comprise a barcode. As noted elsewhere herein, the structure of the barcode may include a number of sequence elements, such as functional sequences 808 (e.g., flow cell ligation sequences, sequencing primer sequences, etc.), barcode sequences 810 (e.g., bead-specific sequences shared by the beads, partition-specific sequences shared by the partitions, etc.), and unique molecular identifiers 812 (e.g., unique sequences within different molecules linked to the beads), or partial sequences thereof. The capture sequence 860 may be configured to be concatenated to a corresponding capture sequence 865. In some cases, the corresponding capture sequence 865 can be coupled to another molecule, which can be an analyte or an intermediate carrier. For example, as shown in fig. 8, a corresponding capture sequence 865 is coupled to a guide RNA molecule 862 comprising a target sequence 864, wherein the target sequence 864 is configured to be linked to an analyte. Another oligonucleotide molecule 807 that is linked to bead 804 comprises a second capture sequence 880 that is configured to be linked to a second corresponding capture sequence 885. As shown in fig. 8, a second corresponding capture sequence 885 is coupled to antibody 882. In some cases, antibody 882 may have binding specificity for an analyte (e.g., a surface protein). Alternatively, antibody 882 may not have binding specificity. Another oligonucleotide molecule 803 attached to the bead 804 comprises a third capture sequence 870 configured to be attached to a second corresponding capture sequence 875. As shown in fig. 8, a third corresponding capture sequence 875 is coupled to molecule 872. The molecule 872 may or may not be configured to target an analyte. The other oligonucleotide molecules 803, 807 may comprise other sequences (e.g., functional sequences, barcode sequences, UMI, etc.) described with respect to the oligonucleotide molecule 805. While fig. 8 shows a single oligonucleotide molecule comprising each capture sequence, it is to be understood that the bead may comprise a set of one or more oligonucleotide molecules for each capture sequence, wherein each oligonucleotide molecule comprises a capture sequence. For example, the beads may comprise any number of sets having one or more different capture sequences. Alternatively or in addition, the beads 804 may comprise other capture sequences. Alternatively or in addition, the beads 804 may contain fewer types of capture sequences (e.g., two capture sequences). Alternatively or in addition, the bead 804 may comprise an oligonucleotide molecule comprising a promoter sequence, such as a specific promoter sequence, such as an mRNA specific promoter sequence (e.g., a poly-T sequence), a targeted promoter sequence, and/or a random promoter sequence, for example, to facilitate determination of gene expression.

Fig. 2 shows an exemplary microfluidic channel structure 200 for generating discrete droplets encapsulating barcode-carrying beads 214 and biological particles 216. Channel structure 200 includes channel segments 201, 202, 204, 206, and 208 that are in fluid communication at channel connection 210. In operation, the channel segment 201 transports an aqueous fluid 212, which may contain a plurality of beads 214 (e.g., gel beads carrying barcode oligonucleotides), along the channel segment 201 into the junction 210. The plurality of beads 214 may be derived from a suspension of beads. For example, channel segment 201 may be connected to a reservoir of an aqueous suspension comprising beads 214. The channel segment 202 conveys an aqueous fluid 212 containing a plurality of biological particles from a biological sample 216 along the channel segment 202 into the junction 210. The plurality of biological particles 216 may be derived from a suspension of a biological sample. For example, the channel segment 202 may be connected to a reservoir of an aqueous suspension containing biological particles 216. In some cases, the aqueous fluid 212 in the first channel segment 201 or the second channel segment 202 or in both segments may contain one or more reagents, as further described elsewhere herein. For example, in some embodiments of the present disclosure, where the biological particles are from immobilized biological samples, the aqueous fluid in the first channel segment and/or the second channel segment delivering the biological samples and beads, respectively, may comprise a unfixed agent. A second fluid 218 that is immiscible with the aqueous fluid 212 is delivered from each of the channel segments 204 and 206 to the connection 210. As the aqueous fluid 212 from each of the channel segments 201 and 202 and the second fluid 218 (e.g., fluorinated oil) from each of the channel segments 204 and 206 meet at the channel connection 210, the aqueous fluid 212 may separate into discrete droplets 220 in the second fluid 218 and flow along the channel segment 208 away from the connection 210. The channel segment 208 may then deliver the discrete droplets of encapsulated biological particles and bar-code-bearing beads to an outlet reservoir fluidly coupled to the channel segment 208, where the discrete droplets may be collected.

Alternatively, the channel segments 201 and 202 may meet at another junction upstream of the junction 210. At such a junction, the beads and biological particles may form a mixture that is directed along another channel to junction 210 to create droplets 220. The mixture may provide beads and biological particles in an alternating manner such that, for example, the droplets comprise a single bead and a single biological particle.

Using such a channel system as illustrated in fig. 2, discrete droplets 220 of individual particles and one bead encapsulating a biological sample may be generated, wherein the bead may carry a barcode and/or another reagent. It is also contemplated that in some cases, the channel system of fig. 2 may be used to generate discrete droplets, where the droplets contain more than one individual biological particle, or do not include a biological sample. Similarly, in some embodiments, the discrete droplets may or may not contain more than one bead. Discrete droplets may also be completely unoccupied (e.g., without beads or biological sample).

In some embodiments, it is desirable that the beads, biological particles from the biological sample, and the discrete droplets generated flow along the channel at a substantially regular flow rate, thereby generating discrete droplets comprising a single bead and a single biological particle. Conventional flow rates and devices that may be used to provide such conventional flow rates are known in the art, see, for example, U.S. patent publication No. 2015/0292988, which is hereby incorporated by reference in its entirety. In some embodiments, the flow rate is set to provide discrete droplets comprising individual beads and biological particles in a yield of greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

E. Support material

Supports (such as beads) that may carry barcodes and/or other reagents may be used in the methods of the present disclosure, and may include, but are not limited to, porous, nonporous, solid, semi-fluid, and/or supports having a combination of these properties. In some embodiments, the support is a bead made of a dissolvable, rupturable, and/or degradable material, such as a gel bead comprising a hydrogel. Alternatively, in some embodiments, the support is non-degradable.

In some embodiments of the present disclosure, the support is a bead that may be encapsulated in discrete droplets with the biological sample. Typically, the beads useful in the embodiments disclosed herein comprise hydrogels. Such gel beads may be formed from molecular precursors, such as polymeric or monomeric materials, that undergo a reaction to form a crosslinked gel polymer. Another semi-solid bead useful in the present disclosure is a liposome bead. In some embodiments, the beads used may be solid beads comprising metals including iron oxide, gold, and silver. In some cases, the beads may be silica beads. In some cases, the beads may be rigid. In other cases, the beads may be flexible and/or compressible. Generally, the beads can have any suitable shape. Examples of bead shapes include, but are not limited to, spherical, non-spherical, oval, oblong, amorphous, circular, cylindrical, and variations thereof.

In some embodiments, multiple beads or a population of beads may be used. The plurality of beads used in these embodiments may be of uniform size, have a relatively monodisperse size distribution, or they may comprise a collection of heterogeneous sizes. In some cases, the bead diameter is at least about 1 micrometer (μm), 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1000 μm (1 mm), or greater. In some cases, the bead diameter may be less than a value of about 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1mm, or less. In some cases, the bead diameter may be in the range of about 40 μm to 75 μm, 30 μm to 75 μm, 20 μm to 75 μm, 40 μm to 85 μm, 40 μm to 95 μm, 20 μm to 100 μm, 10 μm to 100 μm, 1 μm to 100 μm, 20 μm to 250 μm, or 20 μm to 500 μm.

Generally, where it is desired to provide consistent amounts of reagent within discrete droplets, the use of relatively consistent bead characteristics (such as size) provides overall consistency of the contents of each droplet. For example, beads useful in these embodiments of the present disclosure may have the following size distribution: the coefficient of variation of the cross-sectional dimension is less than 50%, less than 40%, less than 30%, less than 20%, and in some cases less than 15%, less than 10%, less than 5%, or less.

Beads useful in the methods of the present disclosure may comprise a range of natural and/or synthetic materials. For example, the beads may comprise natural polymers, synthetic polymers, or both natural and synthetic polymers. Examples of natural polymers include proteins and sugars such as deoxyribonucleic acid, rubber, cellulose, starch (e.g., amylose, amylopectin), proteins, enzymes, polysaccharides, silk, polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan, psyllium, gum arabic, agar, gelatin, shellac, karaya, xanthan, corn gum, guar gum, karaya, agarose, alginic acid, alginate, or natural polymers thereof. Examples of synthetic polymers include acrylic, nylon, silicone, spandex (spandex), viscose rayon, polycarboxylic acid, polyvinyl acetate, polyacrylamide, polyacrylate, polyethylene glycol, polyurethane, polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, polyethylene terephthalate, poly (chlorotrifluoroethylene), poly (ethylene oxide), poly (ethylene terephthalate), polyethylene, polyisobutylene, poly (methyl methacrylate), poly (formaldehyde), polyoxymethylene, polypropylene, polystyrene, poly (tetrafluoroethylene), poly (vinyl acetate), poly (vinyl alcohol), poly (vinyl chloride), poly (vinylidene fluoride), poly (vinyl fluoride), and/or combinations (e.g., copolymers) thereof. The beads may also be formed from materials other than polymers including lipids, micelles, ceramics, glass-ceramics, material composites, metals, other inorganic materials, and the like.

While fig. 1 and 2 have been described in terms of providing discrete droplets that are substantially singly occupied, it is also contemplated in certain embodiments that it is desirable to provide discrete droplets that are multiply occupied, e.g., a single droplet comprising two, three, four or more cells/nuclei from a biological sample, and/or a plurality of different beads, such as beads carrying barcode nucleic acid molecules and/or supports (e.g., beads) carrying reagents such as a unfixed or assay reagent. Thus, as described elsewhere herein, the flow characteristics of the biological particles and/or beads can be controlled to provide such multiple occupied droplets. In particular, the flow parameters of the liquid used in the channel structure may be controlled to provide a given droplet occupancy of greater than about 50%, greater than about 75%, and in some cases greater than about 80%, 90%, 95%, or higher.

In some embodiments, the beads useful in the methods of the present disclosure are supports (e.g., beads) capable of delivering reagents (e.g., a unfixed agent and/or an assay reagent) into the discrete droplets produced comprising biological particles. In some embodiments, different beads (e.g., comprising different reagents) may be introduced from different sources into different inlets leading to a common drop generating junction (e.g., junction 210). In such cases, the flow and frequency of different beads into the channel or junction can be controlled to provide a specific ratio of supports from each source while ensuring that a given pairing or combination of such supports (e.g., beads) enters a partition with a given number of biological particles (e.g., one biological particle and one bead per partition).

The discrete droplets described herein typically have a small volume, for example, less than about 10 microliters (μl), 5 μl, 1 μl, 900 picoliters (pL), 800pL, 700pL, 600pL, 500pL, 400pL, 300pL, 200pL, 100pL, 50pL, 20pL, 10pL, 1pL, 500 nanoliters (nL), 100nL, 50nL, or less. In some embodiments, the total volume of discrete droplets of the resulting encapsulated biological particles is less than about 1000pL, 900pL, 800pL, 700pL, 600pL, 500pL, 400pL, 300pL, 200pL, 100pL, 50pL, 20pL, 10pL, 1pL, or less. It is to be appreciated that the sample fluid volume (e.g., including co-separated biological particles and/or beads) within a droplet can be less than about 90% of the volume, less than about 80% of the volume, less than about 70% of the volume, less than about 60% of the volume, less than about 50% of the volume, less than about 40% of the volume, less than about 30% of the volume, less than about 20% of the volume, or less than about 10% of the volume.

The method of generating discrete droplets that may be used with the methods of the present disclosure is such that a population of discrete droplets or a plurality of discrete droplets comprising biological particles (e.g., biological particles from an immobilized biological sample) and other reagents (e.g., a de-immobilizing agent) are generated. Generally, these methods are easily controlled to provide any suitable number of droplets. For example, at least about 1,000 discrete droplets, at least about 5,000 discrete droplets, at least about 10,000 discrete droplets, at least about 50,000 discrete droplets, at least about 100,000 discrete droplets, at least about 500,000 discrete droplets, at least about 1,000,000 discrete droplets, at least about 5,000,000 discrete droplets, at least about 10,000,000 discrete droplets, or more may be generated or otherwise provided. Further, the plurality of discrete droplets may include both unoccupied droplets and occupied droplets.

In some embodiments of the disclosed methods, discrete droplets of the generated encapsulated biological particles and optionally one or more different beads further comprise other reagents, as described elsewhere herein. In some embodiments, the other reagents encapsulated in the droplet include a lysing agent and/or a deaggregating agent for releasing and/or deaggregating the biomolecular content of the biological particle within the droplet. In some embodiments, the lysing agent and/or the unfixing agent may be contacted with the biological sample suspension at the same time as or immediately prior to introducing the biological particles into the droplet-generating junction (e.g., junction 210) of the microfluidic system. In some embodiments, the agent is introduced through one or more additional channels upstream of the channel connection.

In some embodiments, the biological particles may be co-partitioned along with other reagents. Fig. 3 illustrates one example of a microfluidic channel structure 300 for co-partitioning biological particles and other reagents, including lysing and/or unfixing agents. Channel structure 300 may include channel segments 301, 302, 304, 306, and 308. The channel segments 301 and 302 communicate at a first channel connection 309. The channel segments 302, 304, 306, and 308 communicate at a second channel connection 310. In an exemplary co-partition operation, the channel segment 301 may convey an aqueous fluid 312 containing a plurality of biological particles 314 (e.g., immobilized biological samples) along the channel segment 301 into the second connection 310. Alternatively or additionally, the channel segment 301 may deliver beads (e.g., beads carrying a barcode). For example, the channel segment 301 may be connected to a reservoir of an aqueous suspension comprising biological particles 314. Upstream of and immediately before reaching the second connection 310, the channel segment 301 may meet the channel segment 302 at a first connection 309. The channel segment 302 can transport a plurality of reagents 315 (e.g., lysing or unfixing agents) in the aqueous fluid 312 along the channel segment 302 into the first connection 309. For example, channel segment 302 may be connected to a reservoir containing reagent 315. After the first connection 309, the aqueous fluid 312 in the channel segment 301 may bring both the biological particles 314 and the reagent 315 to the second connection 310. In some cases, the aqueous fluid 312 in the channel segment 301 may include one or more reagents, which may be the same or different reagents than the reagent 315. A second fluid 316 (e.g., fluorinated oil) that is immiscible with the aqueous fluid 312 may be delivered from each of the channel segments 304 and 306 to the second connection 310. As the aqueous fluid 312 from the channel segment 301 and the second fluid 316 from each of the channel segments 304 and 306 meet at the second channel connection 310, the aqueous fluid 312 separates into discrete droplets 318 in the second fluid 316 and flows along the channel segment 308 away from the second connection 310. The channel segment 308 may deliver the discrete droplets 318 to an outlet reservoir fluidly coupled to the channel segment 308, where the discrete droplets may be collected for further analysis.

The discrete droplets generated may contain a single biological particle 314 and/or one or more reagents 315, depending on which reagents are contained in the channel segment 302. In some cases, the discrete droplets generated may further comprise bar code carrying beads (not shown), such as bar code carrying beads that may be added via other channel structures described elsewhere herein. In some cases, the discrete droplets may be unoccupied (e.g., reagent-free, biological particle-free). In general, the channel segments described herein may be coupled to any of a variety of different fluid sources or receiving components, including a reservoir, conduit, manifold, or other system fluid component. It should be understood that the microfluidic channel structure 300 may have other geometries. For example, a microfluidic channel structure may have more than two channel connections. For example, a microfluidic channel structure may have 2, 3, 4, 5 or more channel segments each carrying the same or different types of beads, reagents and/or biological particles, which meet at a channel junction. The fluid flow in each channel segment can be controlled to control the separation of different elements into droplets. Fluid may be directed to flow along one or more channels or reservoirs via one or more fluid flow units. The fluid flow unit may include a compressor (e.g., providing positive pressure), a pump (e.g., providing negative pressure), an actuator, etc., to control the flow of fluid. The fluid may also or alternatively be controlled via an applied pressure differential, centrifugal force, electric pumping, vacuum, capillary or gravity flow, or the like.

Fig. 4 shows an example of a microfluidic channel structure for controlled separation of beads into discrete droplets. The channel structure 400 may include a channel segment 402 that communicates with a reservoir 404 at a channel connection 406 (or intersection). The reservoir 404 may be a chamber. As used herein, any reference to a "reservoir" may also refer to a "chamber. In operation, the aqueous fluid 408 containing the suspended beads 412 may be transported along the channel segment 402 into the connection 406 to encounter the second fluid 410 that is immiscible with the aqueous fluid 408 in the reservoir 404, thereby producing droplets 416, 418 of the aqueous fluid 408 flowing into the reservoir 404. The junction 406 where the aqueous fluid 408 and the second fluid 410 meet may be based on certain geometric parameters (e.g., w, h) such as the hydrodynamic forces at the junction 406, the flow rates of the two fluids 408, 410, the fluid characteristics, and the channel structure 400 ₀ α, etc.) to form droplets. By continuously injecting aqueous fluid 408 from channel segment 402 through connection 406, a plurality of droplets may be collected in reservoir 404.

Fig. 5 shows an example of a microfluidic channel structure for achieving increased droplet generation throughput. The microfluidic channel structure 500 can include a plurality of channel segments 502 and reservoirs 504. Each of the plurality of channel segments 502 may be in fluid communication with a reservoir 504. The channel structure 500 may include a plurality of channel connections 506 between the plurality of channel segments 502 and the reservoir 504. Each channel connection may be a point of droplet generation. The channel segment 402 from the channel structure 400 in fig. 4 and any description of its components may correspond to a given channel segment of the plurality of channel segments 502 in the channel structure 500 and any description of its corresponding components. The repository 404 from the channel structure 400 and any description of its components may correspond to the repository 504 from the channel structure 500 and any description of its corresponding components.

Fig. 6 shows another example of a microfluidic channel structure for achieving increased droplet generation throughput. The microfluidic channel structure 600 may include a plurality of channel segments 602 arranged generally circularly around the perimeter of a reservoir 604. Each of the plurality of channel segments 602 may be in fluid communication with a reservoir 604. The channel structure 600 may include a plurality of channel connections 606 between the plurality of channel segments 602 and the reservoir 604. Each channel connection may be a point of droplet generation. The channel segment 402 from the channel structure 400 in fig. 4 and any description of its components may correspond to a given channel segment of the plurality of channel segments 602 in the channel structure 600 and any description of its corresponding components. The repository 404 from the channel structure 400 and any description of its components may correspond to the repository 604 from the channel structure 600 and any description of its corresponding components. Additional aspects of the microfluidic structures depicted in fig. 4-6, including systems and methods of implementing such microfluidic structures, are provided in U.S. published patent application No. 20190323088, which is incorporated by reference herein in its entirety.

Once the lysing agent and/or the deammobilizing agent are co-segregated in the droplet with the immobilized biological particles, these agents are capable of facilitating release and deammobilization of the biological molecular content of the biological particles within the droplet. As described elsewhere herein, the released unfixed biomolecular content in the droplet remains separate from the content of the other droplets, allowing detection and quantification of the biomolecular analyte of interest present in the different biological sample.

Examples of lysing agents that may be used in the methods of the present disclosure include bioactive agents, e.g., lysing enzymes for lysing different cell types (e.g., gram positive or negative bacteria, plants, yeast, mammals, etc.), such as lysozyme, leucopeptidase, lysostaphin, labase, rhizoctonia lyase (kitalase), lywallase, and a variety of other lysing enzymes available from, e.g., sigma-Aldrich, inc. (St Louis, MO), as well as other commercially available lysing enzymes. Other lysing agents may additionally or alternatively be co-partitioned with the biological sample to cause the contents of the biological sample to be released into the partition. For example, in some cases, surfactant-based lysis solutions may be used to lyse cells or nuclei, but these solutions may be less desirable for emulsion-based systems where surfactants may interfere with stable emulsions. In some embodiments, the lysis solution may comprise a nonionic surfactant, such as Triton X-100 and Tween 20. In some cases, the lysis solution may contain ionic surfactants such as sodium dodecyl sarcosinate and Sodium Dodecyl Sulfate (SDS). Electroporation, thermal, acoustic or mechanical cell disruption may also be used in certain situations, for example non-emulsion based partitioning, such as encapsulation of biological particles, which may be in addition to or instead of droplet partitioning, wherein any pore size of the encapsulate is sufficiently small to retain a nucleic acid fragment of a given size after cell disruption.

In addition to the cleavage and/or unfixed agent being co-partitioned with the biological particle into discrete droplets, it is contemplated that other assay reagents may also be co-partitioned in droplets. For example, deoxyribonuclease and ribonuclease inactivators or inhibitors, such as proteinase K, chelators (such as EDTA), proteases (such as subtilisin a), and other agents for removing or otherwise reducing the negative activity or impact of different cell lysate components on subsequent nucleic acid processing.

In some embodiments, biological particles from a biological sample are provided or encapsulated in discrete partitions (e.g., wells or droplets) with other reagents, and then exposed to an appropriate stimulus to release the biomolecular content of the sample particles and/or the content of a co-partitioned support (e.g., beads). For example, in some embodiments, the chemical stimulus may be co-segregated in the droplet along with the biological particles and the support (e.g., beads, such as gel beads) to allow the support to degrade and release its contents into the droplet. In some embodiments, discrete droplets may be generated with immobilized biological particles and a de-immobilizing agent, where the de-immobilizing agent is contained in a support (e.g., a bead) that can be degraded by thermal stimulation. In such embodiments, the droplets are exposed to a thermal stimulus, thereby degrading the beads and releasing the detackifier. In another embodiment, it is contemplated that the droplet encapsulates immobilized biological particles from an immobilized biological sample, as well as two different beads (e.g., one bead carrying a disarming agent and the other bead carrying an assay reagent), wherein the contents of the two different beads are released by non-overlapping stimuli (e.g., chemical stimulus and thermal stimulus). Such embodiments may allow different reagents to be released into the same discrete droplet at different times. For example, a first bead triggered by a thermal stimulus releases a disaggregating agent into a droplet, and then after a set time, a second bead triggered by a chemical stimulus releases an assay reagent that detects an analyte that disaggregates a biological particle.

Additional assay reagents (such as endonucleases) can also be co-partitioned into discrete droplets with the biological sample to fragment DNA of the biological sample, DNA polymerase and dntps for amplifying nucleic acid fragments of the biological sample, and to attach barcode molecular tags to amplified fragments. Other enzymes may be co-partitioned, including, but not limited to, polymerase, transposase, ligase, protease K, DNA enzyme, subtilisin a, and the like. Additional assay reagents may also include reverse transcriptases, including enzymes, primers, and oligonucleotides having terminal transferase activity, and switch oligonucleotides (also referred to herein as "switch oligonucleotides" or "template switch oligonucleotides") that may be used for template switching.

In some embodiments, template switching may be used to increase the length of the cDNA generated in the assay. In some embodiments, template switching may be used to append a predefined nucleic acid sequence to the cDNA. In the example of template switching, the cDNA may be produced by reverse transcription of a template (e.g., cellular mRNA), where a reverse transcriptase having terminal transferase activity may add additional nucleotides, such as poly-C, to the cDNA in a template-independent manner.

Once the contents of the biological sample cells or nuclei are released into the discrete droplets, the biomolecular components contained therein (e.g., macromolecular components of the biological sample such as RNA, DNA, or proteins) may be further processed within the droplets. According to the methods and systems described herein, the biomolecular content of each biological sample may be provided with a unique barcode identifier, and when the biomolecular components are characterized (e.g., in a sequencing assay), they may be classified as being derived from the same biological sample. By uniquely assigning nucleic acid barcode sequences to individual biological samples or groups of biological samples, the ability to attribute a property to an individual biological sample or group of biological samples will be provided.

In some aspects, the unique identifier barcodes are provided in the form of nucleic acid molecules (e.g., oligonucleotides) that comprise sequences that can be attached to or otherwise associated with the nucleic acid contents of a separate biological sample, or to other components of the biological sample, particularly to fragments of such nucleic acids. In some embodiments, only one nucleic acid barcode sequence is associated with a given discrete droplet, but in some cases, there may be two or more different barcode sequences. The nucleic acid barcode sequence may comprise about 6 to about 20 or more nucleotides within the sequence of a nucleic acid molecule (e.g., an oligonucleotide). In some cases, the barcode sequence may be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or more in length. In some cases, the barcode sequence may be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or more in length. In some cases, the barcode sequence may be up to about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or less in length. These nucleotides may be completely contiguous, i.e. in a single stretch of adjacent nucleotides, or they may be divided into two or more separate subsequences separated by 1 or more nucleotides. In some cases, the separate barcode sequences may be about 4 to about 16 nucleotides in length. In some cases, the barcode sequence may be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode sequence may be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode sequence may be up to about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or less.

In some embodiments, the nucleic acid barcode molecule may also comprise other functional sequences that may be used in the processing of nucleic acids from a biological sample in a droplet. These functional sequences may include, for example, targeting or random/universal amplification primer sequences for amplifying nucleic acid molecules from individual biological samples within a partition, while attaching associated barcode sequences, sequencing primers or primer recognition sites, hybridization sequences or probe sequences, for example, for identifying the presence of these sequences, or for pulling down a barcoded nucleic acid molecule, or any of a number of other potential functional sequences.

In some embodiments, a plurality of nucleic acid barcode molecules (e.g., oligonucleotides) are releasably attached to the beads, wherein all nucleic acid molecules attached to a particular bead will include the same nucleic acid barcode sequence, but represent a plurality of different barcode sequences throughout the population of beads used. In some embodiments, gel beads (e.g., comprising a polyacrylamide polymer matrix) are used as solid supports and delivery vehicles for nucleic acid molecules into droplets, as they are capable of carrying large amounts of nucleic acid molecules, and can be configured to release these nucleic acid molecules upon exposure to a specific stimulus, as described elsewhere herein. In some cases, the bead population provides a diverse barcode sequence library comprising at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences or more.

These nucleic acid barcode molecules may be released from the beads upon application of a specific stimulus to the beads. In some cases, the stimulus may be a light stimulus, for example by cleavage of a photolabile bond, thereby releasing the nucleic acid molecule. In other cases, thermal stimulation may be used, wherein an increase in the temperature of the bead environment will cause cleavage or other release of the bond from the bead. In other cases, chemical stimulus may be used that cleaves the bond of the nucleic acid molecule to the bead, or otherwise causes release of the nucleic acid molecule from the bead. In one instance, such compositions include the polyacrylamide matrices described above for encapsulating biological samples, and can be degraded by exposure to a reducing agent (such as DTT) to release the linked nucleic acid molecules.

F. Use of fixed and unfixed cells from tissue in partition-based assays

As disclosed elsewhere herein, the shredder-immobilization methods of the present disclosure demonstrate that a sample can be obtained by dissociating immobilized cells (e.g., formaldehyde-immobilized biopsy cells) from biological tissue. These immobilized cells may then be provided in discrete partitions (e.g., encapsulated in droplets), optionally as single cells or single nuclei, optionally together with a de-immobilization agent and/or a cryoactive protease capable of reversing immobilization. The optional unfixed agent and/or protease treatment may be used to release and unfixed cellular analytes within a sample (e.g., a cell/cell nucleus, a plurality of cells/cell nuclei, a tissue sample, or other type of biological sample). In some assays, it may be desirable to perform a de-immobilization process to provide cellular analytes for assay that are more closely similar to analytes from fresh samples.

Notably, the shredder-fixing methods of the present disclosure allow fresh tissue samples to be collected, immediately shredded and fixed (e.g., with formaldehyde), after storage for a period of time, subjected to dissociation to obtain fixed cells, or optionally further subjected to a de-fixing treatment, prior to use in a zone-based assay. Thus, it is contemplated that the methods of the present disclosure may be performed as follows: wherein the amount of time before generating the partition containing the sample cells from the tissue is at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 1 week, at least 1 month, at least 6 months or more.

In some aspects, after collecting the tissue sample from the eukaryote, exposing the tissue sample to room temperature or higher for at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days; immediately thereafter, the tissue sample is fixed. In some aspects, the tissue sample is exposed to a temperature of at least about 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, or 90 ℃ for a duration described herein prior to fixation. In some aspects, the tissue sample is exposed to a temperature between: 15 ℃ and 90 ℃, 15 ℃ and 80 ℃, 15 ℃ and 70 ℃, 15 ℃ and 60 ℃, 15 ℃ and 50 ℃, 15 ℃ and 40 ℃, 15 ℃ and 35 ℃, 15 ℃ and 30 ℃, 15 ℃ and 25 ℃, 25 ℃ and 90 ℃, 25 ℃ and 80 ℃, 25 ℃ and 70 ℃, 25 ℃ and 60 ℃, 25 ℃ and 50 ℃, 25 ℃ and 40 ℃, 25 ℃ and 35 ℃, 25 ℃ and 30 ℃, 35 ℃ and 90 ℃, 35 ℃ and 80 ℃, 35 ℃ and 70 ℃, 35 ℃ and 60 ℃, 35 ℃ and 50 ℃, 35 ℃ and 40 ℃, 40 ℃ and 90 ℃, 40 ℃ and 80 ℃, 40 ℃ and 60 ℃, 40 ℃ and 50 ℃.

In another aspect, a fixed tissue sample derived from any one of the time points and/or temperatures described herein can be successfully assayed via the single cell workflow described herein and then returned with viable data. In some aspects, the viable data is not a positive data deletion, but rather successfully elucidates transcriptomes of tissue sample cells. In some aspects, a control tissue sample that is not subjected to fixation, but otherwise is subjected to the same conditions as the tissue sample subjected to fixation, produces in the control tissue sample either infeasible data or a loss of complexity in terms of cell and/or nucleic acid transcript types relative to an experimental tissue sample subjected to fixation.

The present disclosure also provides an assay method comprising the steps of: (a) Generating discrete droplets encapsulating the immobilized cells resulting from the minced-immobilized preparation of biological tissue, and an assay reagent; and (b) detecting the analyte from the reaction of the assay reagent with the immobilized cells. Optionally, the step of the method may further comprise including a disaggregating agent and/or a cryogenically active protease within the discrete droplets generated in step (a), and then detecting in step (b) the analyte from the reaction of the assay reagent with the disaggregated cells.

A wide variety of zone-based assays and systems are known in the art. Assays and systems suitable for use with the present disclosure include, but are not limited to, those described in the following patents: U.S. patent nos. 9694361, 10357771, 10273541 and 10011872, and U.S. published patent application nos. 20180105808, 20190367982 and 20190338353, each of which is incorporated herein by reference in its entirety. It is contemplated that any assay that can be performed using a fresh biological sample, such as single cells/nuclei encapsulated in droplets with barcode-bearing beads, can also be performed using biological samples prepared using the cut-to-fix methods of the present disclosure. That is, in any zone-based assay, dissociated, immobilized cells produced by a chopper-immobilized method can be used, wherein the protocol comprises encapsulating cells from a composition of dissociated, immobilized cells in discrete droplets with an assay reagent, and optionally also with a destabilizing agent and/or a low temperature active protease.

Exemplary assays include single cell transcriptional profiling, single cell sequence analysis, immune profiling of individual T and B cells, single cell/single cell nuclear chromatin accessibility analysis (e.g., ATAC sequence analysis). These exemplary assays can be performed using commercially available systems for encapsulating biological samples, gel beads, barcodes, and/or other compounds/materials in droplets, such as the chromasum system (10X Genomics,Pleasanton,CA,USA).

In some embodiments of the assay method, the discrete droplets further comprise one or more beads. In some embodiments, the beads may comprise an assay reagent and/or a unfixing agent (un-fixing agent). In some embodiments, the barcode is carried by or contained in the bead. Compositions, methods, and systems for sample preparation, amplification, and sequencing of biomolecules from single cells encapsulated in droplets with barcodes are provided, for example, in U.S. patent publication No. 20180216162A1, which is hereby incorporated by reference.

The assay reagents may include reagents for performing one or more additional chemical or biochemical operations on the biological sample encapsulated in the droplet. Thus, assay reagents useful in the assay methods include any reagent useful in performing a reaction such as nucleic acid modification (e.g., ligation, digestion, methylation, random mutagenesis, bisulfite conversion, uracil hydrolysis, nucleic acid repair, capping or uncapping), nucleic acid amplification (e.g., isothermal amplification or PCR), nucleic acid insertion or cleavage (e.g., insertion or cleavage via CRISPR/Cas 9-mediated or transposon-mediated), and/or reverse transcription. In addition, useful assay reagents may include those that allow for the preparation of target sequences or sequencing reads that are specific for a macromolecular component of interest at a higher rate than non-target sequence specific reads.

In addition, the present disclosure provides compositions and systems related to analysis of biological samples prepared from tissue using a cut-to-fix method. In one embodiment, the present disclosure provides a composition comprising a plurality of partitions, wherein a subset of the plurality of partitions comprises dissociated fixed cells or nuclei derived from a chopper-immobilized preparation of biological tissue. In another embodiment, one of the plurality of partitions comprises an immobilized cell/cell nucleus, a destabilizing agent, and a cryoactive protease. In certain embodiments, the fixed cell/nucleus is a single fixed cell/nucleus. In other embodiments, the present disclosure provides compositions comprising a plurality of partitions, wherein the plurality of partitions comprises a plurality of fixed cells or fixed nuclei derived from a chopper-immobilized preparation of a certain biological tissue type. In one embodiment, each of the plurality of partitions further comprises a destabilizing agent and a cryoactive protease, as described herein. The partitions may be droplets or holes.

In some embodiments, one or more of the partitions described herein may further comprise one or more of the following: reverse Transcriptase (RT), beads, and reagents for nucleic acid extension reactions. In at least one embodiment, the protease and/or unfixed agent composition may be provided at a temperature different from ambient temperature. In one embodiment, the temperature is below ambient temperature or above ambient temperature.

As described elsewhere herein, the partitioning method may generate a group of partitions or multiple partitions. In such cases, any suitable number of partitions may be generated or otherwise provided. For example, at least about 1,000 partitions, at least about 5,000 partitions, at least about 10,000 partitions, at least about 50,000 partitions, at least about 100,000 partitions, at least about 500,000 partitions, at least about 1,000,000 partitions, at least about 5,000,000 partitions, at least about 10,000,000 partitions, at least about 50,000,000 partitions, at least about 100,000,000 partitions, at least about 500,000,000 partitions, at least about 1,000,000 partitions, or more partitions may be generated or otherwise provided. Further, the plurality of partitions may include unoccupied partitions (e.g., empty partitions) and occupied partitions. For example, occupied partitions according to the present disclosure comprise immobilized cells/nuclei and, optionally, a deammobilizing agent and/or a cryogenically active protease composition.

In another aspect, the present disclosure relates to a method for separating a plurality of fixed cells or nuclei into individual partitions. In some cases, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10,000, about 15,000, about 20,000, about 25,000, about 30,000, about 35,000, about 40,000, about 50,000, about 60,000, about 70,000, about 80,000, about 90,000, or about 100,000 fixed cells or nuclei may be partitioned into each partition. In some cases, the method further comprises separating from about 50 to about 20,000 immobilized cells or nuclei with each of a plurality of supports comprising adaptors having a barcode sequence, wherein the barcode sequence is unique among each support of the plurality of supports.

Fig. 9 schematically shows an example of a microwell array. The array may be housed within a substrate 900. The substrate 900 includes a plurality of apertures 902. The holes 902 may have any size or shape, and the spacing between holes, the number of holes in each substrate, and the density of holes on the substrate 900 may vary depending on the particular application. In one such example application, the sample molecule 906 may comprise a cell/cell nucleus (e.g., an immobilized cell/cell nucleus or an unfixed cell/cell nucleus) or a cellular component (e.g., a nucleic acid molecule) that is co-partitioned with the bead 904, which may comprise a nucleic acid barcode molecule coupled thereto. The aperture 902 may be loaded using gravity or other loading techniques (e.g., centrifugation, liquid handling, acoustic loading, optoelectronic devices, etc.). In some cases, at least one well 902 contains a single sample molecule 906 (e.g., a cell or nucleus) and a single bead 904.

Reagents may be loaded into the wells sequentially or simultaneously. In some cases, reagents are introduced into the device either before or after a particular operation. In some cases, reagents (which may be provided in droplets or beads in some cases) are introduced sequentially, such that different reactions or manipulations occur at different steps. Reagents (or droplets or beads) may also be loaded at the site of the operation interspersed with reaction or manipulation steps. For example, droplets or beads comprising reagents for fragmenting polynucleotides (e.g., restriction enzymes) and/or other enzymes (e.g., transposases, ligases, polymerases, etc.) can be loaded into one or more wells, followed by loading droplets or beads comprising reagents for ligating nucleic acid barcode molecules to sample nucleic acid molecules. The reagent may be provided simultaneously or sequentially with the sample, wherein the sample is such as a cell/cell nucleus (e.g., immobilized cell/cell nucleus or de-immobilized cell/cell nucleus) or a cellular component (e.g., organelle, protein, nucleic acid molecule, carbohydrate, lipid, etc.). Thus, the use of pores may be useful when performing multi-step operations or reactions.

As described elsewhere herein, nucleic acid barcode molecules and other reagents may be contained within beads or droplets. These beads or droplets may be loaded into the partition (e.g., microwell) before, after, or simultaneously with loading the cells/nuclei (e.g., fixing the cells/nuclei or unfixing the cells/nuclei) such that each cell or nucleus is in contact with a different bead or droplet. This technique can be used to attach unique nucleic acid barcode molecules to nucleic acid molecules obtained from each cell/cell nucleus (e.g., immobilized cell/cell nucleus or de-immobilized cell/cell nucleus). Alternatively or in addition, the sample nucleic acid molecule may be attached to a support. For example, a compartment (e.g., microwell) may comprise a bead having a plurality of nucleic acid barcode molecules coupled thereto. The sample nucleic acid molecule or derivative thereof may be coupled or linked to a nucleic acid barcode molecule on a support. The resulting barcoded nucleic acid molecules can then be removed from the partition and, in some cases, pooled and sequenced. In such cases, the nucleic acid barcode sequence may be used to track the origin of the sample nucleic acid molecule. For example, polynucleotides having the same barcode may be determined to originate from the same cell/nucleus or compartment, while polynucleotides having different barcodes may be determined to originate from different cells/nuclei or compartments.

A variety of methods can be used to load the sample or reagent into the well or microwell. A sample (e.g., a cell, cell nucleus, or cell component) or reagent (as described herein) may be loaded into a well or microwell using external forces (e.g., gravity, electricity, magnetism) or using a mechanism that drives the sample or reagent into the well (e.g., via pressure-driven flow, centrifugation, optoelectronic devices, acoustic loading, electrokinetic pumping, vacuum, capillary flow, etc.). In some cases, a fluid handling system may be used to load a sample or reagent into a well. The loading of the sample or reagent may follow a poisson or non-poisson distribution, such as super-poisson or sub-poisson. The microwell geometry, spacing between wells, density and size can be modified to accommodate useful sample or reagent distributions; for example, the size and spacing of the micro-holes may be adjusted so that the sample or reagent can be distributed in a superpopoison fashion.

In one particular non-limiting example, the microwell array or plate includes pairs of microwells, wherein each pair of microwells is configured to accommodate a droplet (e.g., comprising a single cell/cell nucleus, e.g., a single immobilized cell/cell nucleus or a single disarmed cell/cell nucleus) and a single bead (such as those described herein, which may also be provided in or encapsulated within a droplet in some cases). The droplets and beads (or droplets containing beads) may be loaded simultaneously or sequentially, and the droplets and beads may fuse, for example, when the droplets are in contact with the beads, or when a stimulus (e.g., external force, stirring, heat, light, magnetic force, or electric force, etc.) is applied. In some cases, the loading of droplets and beads is superppoisson. In other examples of microwell pairs, the wells are configured to hold two droplets containing different reagents and/or samples that fuse upon contact or upon application of a stimulus. In such a case, the droplets of one microwell of the pair may contain a reagent that can react with the reagent in the droplets of the other microwell of the pair. For example, one droplet may contain a reagent configured to release a nucleic acid barcode molecule of a bead contained in another droplet located in an adjacent microwell. Upon droplet fusion, the nucleic acid barcode molecules can be released from the beads into the partitions (e.g., microwells or contacted microwell pairs) and can undergo further processing (e.g., barcode addition, nucleic acid reaction, etc.). In the case where cells/nuclei (e.g., fixed cells/nuclei or unfixed cells/nuclei) are loaded in the microwells, one of the droplets may contain reagents for further processing, e.g., lysis reagents for lysing the cells/nuclei upon droplet merger.

The droplets may be separated into one hole. The droplets may be selected or subjected to pre-processing prior to loading into the wells. For example, the droplets may contain cells (e.g., fixed cells or unfixed cells), and only certain droplets, such as those containing a single cell (or at least one cell), may be selected for loading into the well. Such a preselection process may be used to efficiently load individual cells, such as to obtain a non-poisson distribution, or to pre-filter cells with selected characteristics prior to further separation in the well. In addition, the technique may be used to obtain or prevent the formation of cell doublets or multimers prior to or during loading of microwells.

In some cases, the pore may comprise a nucleic acid barcode molecule attached thereto. The nucleic acid barcode molecules may be attached to the surface of the well (e.g., the wall of the well). The nucleic acid barcode molecules of one well (e.g., compartment barcode sequences) may be different from the nucleic acid barcode molecules of another well, which may allow for the identification of the contents contained within a single compartment or well. In some cases, the nucleic acid barcode molecule may comprise a spatial barcode sequence that can identify the spatial coordinates of a well, for example, within a well array or well plate. In some cases, the nucleic acid barcode molecule may include a unique molecular identifier for identifying the individual molecule. In some cases, the nucleic acid barcode molecules may be configured to attach to or capture nucleic acid molecules within a sample or cell/nucleus (e.g., immobilized cell/nucleus or un-immobilized cell/nucleus) distributed in a well. For example, a nucleic acid barcode molecule may comprise a capture sequence that may be used to capture or hybridize to a nucleic acid molecule (e.g., RNA, DNA) within a sample. In some cases, the nucleic acid barcode molecule may be released from the microwell. For example, a nucleic acid barcode molecule may comprise a chemical cross-linker that can be cleaved upon application of a stimulus (e.g., photo-stimulus, magnetic stimulus, chemical stimulus, biological stimulus). The released nucleic acid barcode molecules (which may be hybridized or configured to hybridize to the sample nucleic acid molecules) may be collected and pooled for further processing, which may include nucleic acid processing (e.g., amplification, extension, reverse transcription, etc.) and/or characterization (e.g., sequencing). In such cases, unique compartment barcode sequences can be used to identify the cell/nucleus or compartment from which the nucleic acid molecule originates.

The sample within the well can be characterized. In a non-limiting example, such characterization may include imaging a sample (e.g., a cell nucleus, or a cell component) or derivative thereof. Characterization techniques (such as microscopy or imaging) can be used to measure the profile of the sample in a fixed spatial location. For example, when cells/nuclei (e.g., fixed cells/nuclei or unfixed cells/nuclei) are separated, optionally together with beads, imaging each microwell and the contents contained therein can provide useful information about: cell/nucleus duplex formation (e.g., frequency, spatial location, etc.), cell-bead pair efficiency, cell viability, cell size, cell morphology, expression level of biomarkers (e.g., surface markers, molecules fluorescently labeled therein, etc.), cell or bead loading rate, number of cell-bead pairs, cell-cell interactions (when two or more cells are co-separated). Alternatively or in addition, imaging may be used to characterize the amount of amplification product in a well.

In operation, the wells may be loaded with sample and reagent simultaneously or sequentially. When loading cells/nuclei (e.g., fixing cells/nuclei or unfixing cells/nuclei), the wells may be washed, e.g., to remove excess cells/nuclei from the wells, microwell arrays or plates. Similarly, a wash may be performed to remove excess beads or other reagents from the wells, microwell arrays or microwell plates. In addition, the cells/nuclei may be lysed in the various partitions to release intracellular/nuclear components or cellular analytes. Alternatively, the cells/nuclei may be immobilized or permeabilized in the various partitions. The intracellular components or cellular analytes may be coupled to the support, e.g., on the surface of a microwell, on a solid support (e.g., a bead), or they may be collected for further downstream processing. For example, after cell/nucleus lysis, intracellular/nuclear components or cellular analytes may be transferred to individual droplets or other partitions for barcode addition. Alternatively or in addition, an intracellular/nuclear component or cellular analyte (e.g., a nucleic acid molecule) may be coupled to a bead comprising a nucleic acid barcode molecule; the beads may then be collected and further processed, e.g., subjected to a nucleic acid reaction such as reverse transcription, amplification or extension, and the nucleic acid molecules thereon may be further characterized, e.g., via sequencing. Alternatively, or in addition, the intracellular/nuclear component or the cellular analyte may be barcoded in the well (e.g., using beads comprising releasable nucleic acid barcode molecules, or on the surface of microwells comprising nucleic acid barcode molecules). The barcoded nucleic acid molecules or analytes may be further processed in the well or the barcoded nucleic acid molecules or analytes may be collected from the individual partitions and subjected to further processing outside the partitions. Further processing may include nucleic acid processing (e.g., performing amplification, extension) or characterization (e.g., fluorescent monitoring, sequencing of amplified molecules). In any convenient or useful step, the wells (or microwell array or microwell plate) may be sealed (e.g., using oil, film, wax, etc.), which enables storage of the assay or selective introduction of additional reagents.

Once sealed, the well may be subjected to conditions that further process the cell/nucleus (or cells/nuclei) in the well. For example, the reagents in the wells may allow for further processing of the cells/nuclei, such as cell lysis, as described further herein. Alternatively, the well (or wells, such as those in a well-based array) containing the cell/nucleus (or multiple cells/nuclei) may be subjected to a freeze-thaw cycle to process the cell/nucleus (or multiple cells/nuclei), e.g., cell lysis. The cell-loaded wells may be subjected to a freezing temperature (e.g., 0 ℃, less than 0 ℃, -5 ℃, -10 ℃, -15 ℃, -20 ℃, -25 ℃, -30 ℃, -35 ℃, -40 ℃, -45 °, -55 ℃, -60 ℃, -65 ℃, -70 ℃, -80 ℃, or-85 ℃). Freezing can be performed in a suitable manner, such as a sub-zero freezer or a dry ice/ethanol bath. After initial freezing, the well (or wells) containing the cell/nucleus (or cells/nuclei) may be subjected to a freeze-thaw cycle to lyse the cell/nucleus (or cells/nuclei). In one embodiment, the initially frozen well (or wells) is thawed to a temperature above the freezing temperature (e.g., room temperature or 25 ℃). In another embodiment, the freezing time is less than 10 minutes (e.g., 5 minutes or 7 minutes), followed by thawing time at room temperature is less than 10 minutes (e.g., 5 minutes or 7 minutes). The freeze-thaw cycle may be repeated multiple times, for example 2, 3, or 4 times, to obtain lysis of the cell/nucleus (or cells/nuclei) in the well (or wells). In one embodiment, the freezing, thawing, and/or freeze/thaw cycles are performed without lysis buffer.

FIG. 10 schematically shows an example workflow for processing nucleic acid molecules within a sample. A substrate 1000 including a plurality of micro-holes 1002 may be provided. The sample 1006 may comprise cells/nuclei (e.g., immobilized cells/nuclei or de-immobilized cells/nuclei), cellular components, or analytes (e.g., proteins and/or nucleic acid molecules), which may be co-partitioned in a plurality of microwells 1002 with a plurality of beads 1004 comprising nucleic acid barcode molecules. During process 1010, sample 1006 may be processed within the partition. For example, the cells may be subjected to conditions sufficient to lyse the cells/nuclei (e.g., fixed cells/nuclei or unfixed cells/nuclei) and release the analytes contained therein. In process 1020, the beads 1004 may be further processed. For example, processes 1020a and 1020b schematically show different workflows depending on the characteristics of the beads 1004.

In 1020a, the bead includes a nucleic acid barcode molecule attached thereto, and a sample nucleic acid molecule (e.g., RNA, DNA) can be attached to the nucleic acid barcode molecule, e.g., via ligation hybridization. This attachment may occur on the beads. In process 1030, beads 1004 may be collected from multiple wells 1002 and combined. Further processing may be performed in process 1040. For example, one or more nucleic acid reactions, such as reverse transcription, nucleic acid extension, amplification, ligation, transposition, and the like, may be performed. In some cases, the adapter sequence is linked to a nucleic acid molecule or derivative thereof, as described elsewhere herein. For example, sequencing primer sequences may be added to each end of the nucleic acid molecule. In process 1050, further characterization (such as sequencing) may be performed to generate sequencing reads. These sequencing reads can yield information about individual cells/nuclei or cell/nucleus populations (e.g., fixed cells/nuclei or unfixed cells/nuclei), which can be presented visually or graphically, for example, in fig. 1055.

In 1020b, the bead comprises a nucleic acid barcode molecule releasably attached thereto, as described below. The beads may degrade or otherwise release the nucleic acid barcode molecules into the wells 1002; the nucleic acid barcode molecules can then be used to barcode the nucleic acid molecules within the well 1002. Further processing may be performed either inside the partition or outside the partition. For example, one or more nucleic acid reactions, such as reverse transcription, nucleic acid extension, amplification, ligation, transposition, and the like, may be performed. In some cases, the adapter sequence is linked to a nucleic acid molecule or derivative thereof, as described elsewhere herein. For example, sequencing primer sequences may be added to each end of the nucleic acid molecule. In process 1050, further characterization (such as sequencing) may be performed to generate sequencing reads. These sequencing reads can yield information about individual cells/nuclei or cell/nucleus populations (e.g., fixed cells/nuclei or unfixed cells/nuclei), which can be presented visually or graphically, for example, in fig. 1055.

G. Additional method

The present disclosure provides methods and systems for multiplexing samples (e.g., cells/nuclei, fixed cells/nuclei, or unfixed cells/nuclei) and otherwise increasing sample throughput for analysis. For example, a single or integrated process workflow may allow for processing, identifying, and/or analyzing more or multiple analytes, more or multiple types of analytes, and/or more or multiple types of analyte characterization. For example, in the methods and systems described herein, one or more labeling agents capable of binding to or otherwise coupling to one or more cells/nuclei (e.g., cells/nuclei, immobilized cells/nuclei, or unfixed cells/nuclei) or cell/nucleus features can be used to characterize the cells/nuclei and/or the cell/nucleus features. In some cases, the cell features comprise cell surface features and the nuclear features comprise nuclear membrane features. Cell surface or nuclear membrane protein characteristics may include, but are not limited to, receptors, antigens, surface proteins, transmembrane proteins, clusters of differentiated proteins, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, cell-cell interactions, protein complexes, antigen presenting complexes, major histocompatibility complexes, engineered T cell receptors, B cell receptors, chimeric antigen receptors, gap junctions and adhesion junctions, or any combination thereof. In some cases, the cell/nuclear characteristics may include intracellular/nuclear analytes, such as proteins, protein modifications (e.g., phosphorylation states or other post-translational modifications), nuclear proteins, nuclear membrane proteins, or any combination thereof. The labeling agent may include, but is not limited to, proteins, peptides, antibodies (or epitope-binding fragments thereof), lipophilic moieties (such as cholesterol), cell surface receptor binding molecules, receptor ligands, small molecules, bispecific antibodies, bispecific T cell adaptors, T cell receptor adaptors, B cell receptor adaptors, antibody prodrugs, aptamers, monoclonal antibodies, affimer, darpin, and protein scaffolds, or any combination thereof. The labeling agent may include (e.g., be linked to) a reporter oligonucleotide that indicates the cell surface/nuclear membrane protein characteristics to which the binding group binds. For example, the reporter oligonucleotide may comprise a barcode sequence (e.g., a reporter sequence) that allows for identification of the marker agent. For example, a marker specific for one type of cell/nuclear feature (e.g., a first cell surface feature or a first nuclear membrane feature) may have a first reporter oligonucleotide coupled thereto, while a marker specific for a different cell/nuclear feature (e.g., a second cell surface feature or a first nuclear membrane feature) may have a different reporter oligonucleotide coupled thereto. For a description of exemplary labeling agents, reporter oligonucleotides and methods of use, see, e.g., U.S. patent 10,550,429; U.S. patent publication 20190177800; and U.S. patent publication 20190367969, each of which is incorporated by reference herein in its entirety for all purposes.

In one particular example, a library of potential cell/nuclear signature markers may be provided, wherein the respective cell/nuclear signature markers are associated with a nucleic acid reporter such that a different reporter oligonucleotide sequence is associated with each marker that is capable of binding to a particular cell/nuclear signature. In other aspects, different members of the library can be characterized by the presence of different oligonucleotide sequence tags. For example, an antibody capable of binding to a first protein may have a first reporter oligonucleotide sequence associated therewith, while an antibody capable of binding to a second protein may have a different reporter oligonucleotide sequence associated therewith. The presence of a particular oligonucleotide sequence may be indicative of the presence of a particular antibody or cell/nuclear feature that may be recognized or bound by a particular antibody.

For workflows involving the use of fixatives and/or unfixed agents, the labeling agent can be used to label the sample at different points in time (e.g., cell/cell nucleus, fixed cell/cell nucleus, or unfixed cell/cell nucleus). In one embodiment, a plurality of cells/nuclei are labeled before and/or after treatment with the fixative. In another embodiment, a plurality of fixed cells/nuclei are labeled before treatment with the unfixed agent and/or after treatment with the unfixed agent. In a further embodiment, a plurality of disarmed cells/nuclei are labeled before being separated into partitions (e.g., wells or droplets) for further processing. In another embodiment, the methods, compositions, systems, and kits described herein provide labeled cells/nuclei, labeled fixed cells/nuclei, or labeled unfixed cells/nuclei.

A labeling agent capable of binding or otherwise coupling to one or more cells/nuclei may be used to characterize the cells/nuclei as belonging to a particular cell/nucleus group. For example, a labeling agent may be used to label a sample of cells/nuclei or a group of cells/nuclei. In this way, one set of cells/nuclei may be labeled differently than another set of cells/nuclei. In one example, a first set of cells/nuclei may be derived from a first sample and a second set of cells/nuclei may be derived from a second sample. The tagging agent may allow the first and second sets to have different tagging agents (or reporter oligonucleotides associated with the tagging agents). This may, for example, facilitate multiplexing, wherein the cells/nuclei of the first group and the cells/nuclei of the second group may be labeled separately and then pooled together for downstream analysis. Downstream detection of the tag may indicate that the analyte belongs to a particular group.

For example, the reporter oligonucleotide may be linked to an antibody or epitope-binding fragment thereof, and labeling the cell/cell nucleus may include subjecting the antibody-linked barcode molecule or epitope-binding fragment-linked barcode molecule to conditions suitable for binding the antibody to a molecule present on the surface of the cell/cell nucleus. The binding affinity between the antibody or epitope-binding fragment thereof and the molecule present on the surface may be within a desired range to ensure that the antibody or epitope-binding fragment thereof remains bound to the molecule. For example, the binding affinity may be within a desired range to ensure that the antibody or epitope-binding fragment thereof remains bound to the molecule during various sample processing steps (e.g., partitioning and/or nucleic acid amplification or extension). The dissociation constant (Kd) between the antibody or epitope-binding fragment thereof and the molecule to which it binds may be less than about 100. Mu.M, 90. Mu.M, 80. Mu.M, 70. Mu.M, 60. Mu.M, 50. Mu.M, 40. Mu.M, 30. Mu.M, 20. Mu.M, 10. Mu.M, 9. Mu.M, 8. Mu.M, 7. Mu.M, 6. Mu.M, 5. Mu.M, 4. Mu.M, 3. Mu.M, 2. Mu.M, 1. Mu.M, 900nM, 800nM, 700nM, 600nM, 500nM, 400nM, 300nM, 200nM, 100nM, 90nM, 80nM, 70nM, 60nM, 50nM, 40nM, 30nM, 20nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 900pM, 800pM, 700pM, 600pM, 500pM, 400pM, 90pM, 80nM, 70nM, 60nM, 50nM, 40nM, 10nM, 4 pM. For example, the dissociation constant may be less than about 10 μm.

In another example, the reporter oligonucleotide may be coupled to a Cell Penetrating Peptide (CPP), and labeling the cell/nucleus may include delivering the CPP-coupled reporter oligonucleotide into an analyte carrier. Labeling the analyte carrier can include delivering the CPP-conjugated oligonucleotide into a cell or nucleus via a cell penetrating peptide. CPPs that can be used in the methods provided herein can comprise at least one nonfunctional cysteine residue, which can be free or derivatized, for disulfide bond formation with an oligonucleotide that has been modified for such bond. Non-limiting examples of CPPs that may be used in the embodiments herein include permeants, transporters, plsl, TAT (48-60), pVEC, MTS, and MAP. The cell penetrating peptide useful in the methods provided herein may have the ability to induce cell penetration of at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the cells/nuclei of the cell/nucleus population. CPP can be an arginine-rich peptide transporter. CPP may be a permeant or Tat peptide. In another example, the reporter oligonucleotide may be coupled to a fluorophore or dye and labeling the cell/nucleus may include subjecting the fluorophore-linked barcode molecule to conditions suitable for binding the fluorophore to the cell surface or nuclear membrane. In some cases, the fluorophore can interact strongly with the lipid bilayer, and labeling the cell/cell nucleus can include subjecting the fluorophore-linked barcode molecule to conditions such that the fluorophore binds to or intercalates into the membrane of the cell membrane or cell nucleus. In some cases, the fluorophore is a water-soluble organic fluorophore. In some cases, the fluorophore is Alexa 532 maleimide, tetramethylrhodamine-5-maleimide (TMR maleimide), BODIPY-TMR maleimide, sulfo-Cy 3 maleimide, alexa 546 carboxylic acid/succinimidyl ester, atto 550 maleimide, cy3 carboxylic acid/succinimidyl ester, cy3B carboxylic acid/succinimidyl ester, atto 565 biotin, sulforhodamine B, alexa 594 maleimide, texas Red maleimide, alexa 633 maleimide, abberior STAR 635P azide, atto 647N maleimide, atto 647SE, or sulfo-Cy 5 maleimide. See, for example, hughes L D et al, PLoS one.2014, month 2, 4; 9 (2) e87649, which is hereby incorporated by reference in its entirety for all purposes, is intended to illustrate organic fluorophores.

The reporter oligonucleotide may be coupled to a lipophilic molecule and labeling the cell/nucleus may include delivering the nucleic acid barcode molecule to a cell membrane or nuclear membrane by the lipophilic molecule. The lipophilic molecules may be associated with and/or intercalated into lipid membranes, such as cell membranes and nuclear membranes. In some cases, the insertion may be reversible. In some cases, the binding between the lipophilic molecule and the cell membrane or nuclear membrane may be such that the membrane retains the lipophilic molecule (e.g., and its bound components, such as a nucleic acid barcode molecule) during subsequent processing (e.g., partitioning, cell permeabilization, amplification, pooling, etc.). The reporter nucleotide may enter the intracellular space and/or nucleus. In one embodiment, the reporter oligonucleotide coupled to the lipophilic molecule will remain associated with and/or inserted into the lipid membrane (as described herein) via the lipophilic molecule until lysis of the cell/nucleus occurs, e.g., within the partition.

The reporter oligonucleotide may be part of a nucleic acid molecule comprising any number of functional sequences as described elsewhere herein, such as a target capture sequence, a random primer sequence, etc., and coupled to another nucleic acid molecule that is or is derived from an analyte.

Prior to partitioning, the cells/nuclei may be incubated with a library of labeling agents, which may be labeling agents for a wide variety of different cell/nuclear characteristics (e.g., receptors, proteins, etc.), and include their associated reporter oligonucleotides. Unbound labeling agent can be washed away from cells/nuclei, which can then be co-partitioned (e.g., co-partitioned into droplets or wells) along with partition-specific barcode oligonucleotides (e.g., attached to a support such as a bead or gel bead) as described elsewhere herein. Thus, a partition may include one or more cells/nuclei as well as bound labeling agents and their known associated reporter oligonucleotides.

In other cases, for example to facilitate sample multiplexing, a labeling agent specific for a particular cell/nuclear feature may have a first plurality of labeling agents (e.g., antibodies or lipophilic moieties) coupled to a first reporter oligonucleotide and a second plurality of labeling agents coupled to a second reporter oligonucleotide. For example, the first plurality of labeling agents and the second plurality of labeling agents may interact with different cells/nuclei, cell/nucleus populations, or samples, thereby allowing a particular reporter oligonucleotide to indicate a particular cell/nucleus population (or cell/nucleus or sample) and cell/nucleus characteristics. In this way, different samples or groups may be processed independently and then combined together for pooled analysis (e.g., partition-based bar codes as described elsewhere herein). See, for example, U.S. patent publication 20190323088, which is hereby incorporated by reference in its entirety for all purposes.

As described elsewhere herein, a library of markers can be associated with a particular cell/nucleus characteristic, and can also be used to identify that an analyte originates from a particular cell/nucleus population or sample. The cell/nucleus population may be incubated with multiple libraries such that one or more cells/nuclei contain multiple labeling agents. For example, the cell/nucleus may comprise a lipophilic labelling agent and an antibody coupled thereto. The lipophilic labelling agent may indicate that the cell/cell nucleus is a member of a particular cell sample, and the antibody may indicate that the cell/cell nucleus contains a particular analyte. In this way, the reporter oligonucleotide and the labeling agent may allow for multiplex analysis of multiple analytes.

In some cases, these reporter oligonucleotides may comprise a nucleic acid barcode sequence that: these nucleic acid barcode sequences allow the identification of the labeling agent to which the reporter oligonucleotide is coupled. The use of oligonucleotides as reporters may provide the following advantages: can create significant diversity in sequence while also being readily attachable to most biomolecules (e.g., antibodies, etc.), and easy to detect (e.g., using sequencing or array techniques).

The attachment (coupling) of the reporter oligonucleotide to the labeling agent may be accomplished by any of a variety of direct or indirect, covalent or non-covalent associations or linkages. For example, the oligonucleotide may be conjugated using chemical conjugation techniques (e.g., lightning available from Innova Biosciences) Antibody labeling kit) is covalently linked to a labelA portion of an agent (such as a protein, e.g., an antibody or antibody fragment), and using other non-covalent attachment mechanisms, e.g., using biotinylated antibodies and oligonucleotides with avidin or streptavidin linkers (or beads comprising one or more biotinylated linkers coupled to the oligonucleotides). Antibodies and oligonucleotide biotinylation techniques are available. See, e.g., fang et al, "Fluoride-Cleavable Biotinylation Phosphoramidite for 5' -end-Labelling and Affinity Purification of Synthetic Oligonucleotides," Nucleic Acids res.2003, 1 month 15; 31 708-715, which are incorporated herein by reference in their entirety for all purposes. Also, protein and peptide biotinylation techniques have been developed and are ready for use. See, for example, U.S. patent No. 6,265,552, which is incorporated by reference herein in its entirety for all purposes. In addition, click chemistry such as methyltetrazine-PEG 5-NHS ester reaction, TCO-PEG4-NHS ester reaction, and the like can be used to couple the reporter oligonucleotide to the labeling agent. Commercially available kits (such as those from thunder and Abcam) may be used to couple the reporter oligonucleotide to the labeling agent as appropriate. In another example, the labeling agent is coupled indirectly (e.g., via hybridization) to a reporter oligonucleotide that comprises a barcode sequence that identifies the labeling agent. For example, the labeling agent can be directly coupled (e.g., covalently bound) to a hybridization oligonucleotide comprising a sequence that hybridizes to a sequence of the reporter oligonucleotide. Hybridization of the hybridization oligonucleotide to the reporter oligonucleotide couples the labeling agent to the reporter oligonucleotide. In some embodiments, the reporter oligonucleotide may be released from the tagging agent, such as upon application of a stimulus. For example, the reporter oligonucleotide may be linked to the labeling agent by an labile bond (e.g., chemically labile, photolabile, thermally labile, etc.), as generally described elsewhere herein for release of molecules from the support. In some cases, the reporter oligonucleotides described herein may include one or more functional sequences useful for subsequent processing, such as an adapter sequence, a Unique Molecular Identifier (UMI) sequence, a sequencer-specific flow cell ligation sequence (such as P5, P7 Or a portion of the P5 or P7 sequence), a primer or primer binding sequence, a sequencing primer or primer binding sequence (such as R1, R2 or a portion of the R1 or R2 sequence).

In some cases, the labeling agent may comprise a reporter oligonucleotide and a tag. The label may be a fluorophore, a radioisotope, a molecule capable of undergoing a colorimetric reaction, a magnetic particle, or any other suitable molecule or compound capable of detection. The tag may be conjugated directly or indirectly to a labeling agent (or reporter oligonucleotide) (or the tag may be conjugated to a molecule that can bind to a labeling agent or reporter oligonucleotide). In some cases, the tag is conjugated to an oligonucleotide that is complementary to the sequence of the reporter oligonucleotide, and the oligonucleotide may be allowed to hybridize to the reporter oligonucleotide.

FIG. 11 depicts an exemplary labeling agent (1110, 1120, 1130) comprising a reporter oligonucleotide (1140) attached thereto. The labeling agent 1110 (e.g., any of the labeling agents described herein) is attached (either directly (e.g., covalently) or indirectly) to the reporter oligonucleotide 1140. Reporter oligonucleotide 1140 may comprise barcode sequence 1142 identifying marker 1110. Reporter oligonucleotide 1140 may also comprise one or more functional sequences 1143 useful for subsequent processing, such as an adapter sequence, a Unique Molecular Identifier (UMI) sequence, a sequencer-specific flow cell ligation sequence (such as P5, P7 or partial P5 or P7 sequences), a primer or primer binding sequence, a sequencing primer or primer binding sequence (such as R1, R2 or partial R1 or R2 sequences).

Referring to fig. 11, in some cases, reporter oligonucleotide 1140 conjugated to a labeling agent (e.g., 1110, 1120, 1130) comprises a primer sequence 1141, a barcode sequence 1142 identifying the labeling agent (e.g., 1110, 1120, 1130), and a functional sequence 1143. Functional sequence 1143 may be configured to hybridize to complementary sequences, such as those present on nucleic acid barcode molecule 1190 (not shown), such as those described elsewhere herein. In some cases, nucleic acid barcode molecules 1190 are attached to a support (e.g., a bead, such as a gel bead), such as those described elsewhere herein. For example, nucleic acid barcode molecule 1190 may be attached to a support via releasable bonds (e.g., including labile bonds), such as those described elsewhere herein. In some cases, reporter oligonucleotide 1140 comprises one or more additional functional sequences, such as those described above.

In some cases, the tagging agent 1110 is a protein or polypeptide (e.g., an antigen or a desired antigen) comprising a reporter oligonucleotide 1140. In some cases, the tagging agent 1110 is an antibody 1120 (or epitope binding fragment thereof) comprising the tagging oligonucleotide 1140, wherein the tagging agent 1110 is a lipophilic moiety (e.g., cholesterol) comprising the tagging oligonucleotide 1140, wherein the lipophilic moiety is selected such that the tagging agent 1110 is integrated into a cell membrane or nucleus, the tagging oligonucleotide 1140 comprises a barcode sequence 1142 identifying the lipophilic moiety 1110, which in some cases is used to tag a cell/nucleus (e.g., a cell/cell nucleus, a cell sample, etc.) and may be used to perform multiplex assays as described elsewhere herein, in some cases the tagging agent is an antibody 1120 (or epitope binding fragment thereof) comprising the tagging oligonucleotide 1140, the tagging oligonucleotide 1140 comprises a barcode sequence 1142, which in some cases is used to identify the presence of a target (i.e., a molecule or compound to which the antibody 1120 binds) such as the antibody 1120, in an embodiment 1132 has a tag 1132, which in some cases is used to tag 1132, such as a binding peptide 1133, a binding molecule 1132, or a binding molecule 1133, such as a binding molecule 1133, may be used to a binding molecule 1133, or a binding molecule 1132, such as a binding molecule 1133, the labeling agent 1130 comprises a plurality of MHC molecules (e.g., MHC multimers, which may be coupled to a support (e.g., 1133)). There are many possible configurations of class I and/or class II MHC multimers that can be used with the compositions, methods, and systems disclosed herein Types, e.g. MHC tetramers, MHC pentamers (MHC assembled via coiled-coil domains, e.gMHC class I pentamer (promimune, ltd.)), MHC octamer, MHC dodecamer, MHC-decorated dextran molecules (e.g., MHC +.>(Immudex)), and the like. For a description of exemplary labeling agents (including antibody and MHC-based labeling agents), reporter oligonucleotides, and methods of use, see, e.g., U.S. patent 10,550,429 and U.S. patent publication 20190367969, each of which is incorporated by reference herein in its entirety for all purposes.

Fig. 12 shows another example of a bead carrying a bar code. In some embodiments, the analysis of multiple analytes (e.g., RNA and one or more analytes, using the labeling agents described herein) can include a nucleic acid barcode molecule, as generally depicted in fig. 12. In some embodiments, nucleic acid barcode molecules 1210 and 1212 are attached to support 1230 via releasable bonds 1240 (e.g., including labile bonds) as described elsewhere herein. The nucleic acid barcode molecule 1210 may comprise an adaptor sequence 1211, a barcode sequence 1212, and an adaptor sequence 1213. The nucleic acid barcode molecule 1220 can comprise an adaptor sequence 1221, a barcode sequence 1212, and an adaptor sequence 1223, wherein the adaptor sequence 1223 comprises a different sequence than the adaptor sequence 1213. In some cases, the adapter 1211 and the adapter 1221 comprise the same sequence. In some cases, the adapter 1211 and the adapter 1221 comprise different sequences. Although support 1230 is shown as comprising nucleic acid barcode molecules 1210 and 1220, any suitable number of barcode molecules comprising common barcode sequence 1212 are contemplated herein. For example, in some embodiments, support 1230 further comprises nucleic acid barcode molecules 1250. Nucleic acid barcode molecule 1250 can comprise an adaptor sequence 1251, a barcode sequence 1212, and an adaptor sequence 1253, wherein adaptor sequence 1253 comprises a sequence different from adaptor sequences 1213 and 1223. In some cases, the nucleic acid barcode molecule (e.g., 1210, 1220, 1250) comprises one or more additional functional sequences, such as UMI or other sequences described herein. The nucleic acid barcode molecule 1210, 1220 or 1250 may interact with an analyte as described elsewhere herein, e.g., as depicted in fig. 13A-13C.

Referring to FIG. 13A, in the case of labeling cells/nuclei with a labeling agent, sequence 1323 may be complementary to the adaptor sequence of the reporter oligonucleotide. The cells/nuclei can be contacted with one or more reporter oligonucleotide 1310 conjugated labeling agents 1320 (e.g., polypeptides, antibodies, or other labeling agents described elsewhere herein). In some cases, the cells/nuclei may be further processed prior to bar coding. For example, such processing steps may include one or more washing steps and/or cell sorting steps. In some cases, cells/nuclei bound to a label 1320 conjugated to an oligonucleotide 1310 and a support 1330 (e.g., a bead, such as a gel bead) comprising a nucleic acid barcode molecule 1390 are partitioned into one of a plurality of partitions (e.g., a droplet of a droplet emulsion or a well of a microwell array). In some cases, the partition contains at most a single cell/nucleus that binds to the labeling agent 1320. In some cases, the reporter oligonucleotide 1310 conjugated to a labeling agent 1320 (e.g., a polypeptide, antibody, pMHC molecule such as MHC multimer, etc.) comprises a first adapter sequence 1311 (e.g., a primer sequence), a barcode sequence 1312 identifying the labeling agent 1320 (e.g., a peptide of a polypeptide, antibody, or pMHC molecule or complex), and an adapter sequence 1313. The adaptor sequence 1313 may be configured to hybridize to a complementary sequence, such as sequence 1323 present on the nucleic acid barcode molecule 1390. In some cases, oligonucleotide 1310 comprises one or more additional functional sequences, such as those described elsewhere herein.

The barcoded nucleic acids may be generated from the constructs described in figures 13A-13C (e.g., via a nucleic acid reaction, such as nucleic acid extension or ligation). For example, sequence 1313 can then be hybridized with complementary sequence 1323 to produce (e.g., via a nucleic acid reaction, such as nucleic acid extension or ligation) a barcoded nucleic acid molecule comprising cell/cell-core (e.g., partition-specific) barcode sequence 1321 (or its reverse complement) and reporter sequence 1312 (or its reverse complement). The barcoded nucleic acid molecules can then optionally be processed as described elsewhere herein, for example, to amplify the molecule and/or to supplement the sequencing platform specific sequences to the fragments. See, for example, U.S. patent publication 2018/0105808, which is hereby incorporated by reference in its entirety for all purposes. The barcoded nucleic acid molecules or derivatives generated therefrom can then be sequenced on a suitable sequencing platform.

In some embodiments, multiple analytes (e.g., a nucleic acid and one or more analytes, using a labeling agent as described herein) can be analyzed. For example, the workflow may include the workflow generally depicted in any of fig. 13A-13C, or a combination of workflows for individual analytes as described elsewhere herein. For example, multiple analytes may be analyzed using a combination of the workflows generally depicted in fig. 13A-13C.

In some cases, analysis of analytes (e.g., nucleic acids, polypeptides, carbohydrates, lipids, etc.) includes the workflow generally depicted in fig. 13A. Nucleic acid barcode molecule 1390 may be co-partitioned with one or more analytes. In some cases, nucleic acid barcode molecules 1390 are attached to a support 1330 (e.g., a bead, such as a gel bead), such as those described elsewhere herein. For example, nucleic acid barcode molecule 1390 may be attached to support 1330 via releasable bond 1340 (e.g., comprising an labile bond), such as those described elsewhere herein. Nucleic acid barcode molecule 1390 may comprise barcode sequence 1321, and optionally comprise other additional sequences, such as UMI sequence 1322 (or other functional sequences described elsewhere herein). Nucleic acid barcode molecule 1390 may comprise sequence 1323, which may be complementary to another nucleic acid sequence such that it can hybridize to a particular sequence.

For example, sequence 1323 may comprise a poly-T sequence and may be used to hybridize to mRNA. Referring to fig. 13C, in some embodiments, nucleic acid barcode molecule 1390 comprises sequence 1323 that is complementary to sequence of RNA molecule 1360 from a cell/nucleus. In some cases, sequence 1323 comprises a sequence specific for an RNA molecule. Sequence 1323 may comprise a known sequence or a targeting sequence, or a random sequence. In some cases, a nucleic acid extension reaction may be performed, resulting in a barcoded nucleic acid product comprising sequence 1323, barcode sequence 1321, UMI sequence 1322, any other functional sequences, and a sequence corresponding to RNA molecule 1360.

In another example, sequence 1323 may be complementary to an overhang sequence or an adapter sequence that has been added to the analyte. For example, referring to fig. 13B, in some embodiments, primer 1350 comprises a sequence complementary to a sequence of a nucleic acid molecule 1360 (such as an RNA encoding a BCR sequence) from an analyte carrier. In some cases, primer 1350 comprises one or more sequences 1351 that are not complementary to RNA molecule 1360. Sequence 1351 may be a functional sequence as described elsewhere herein, e.g., an adapter sequence, a sequencing primer sequence, or a sequence that facilitates coupling to a flow cell of a sequencer. In some cases, primer 1350 comprises a poly T sequence. In some cases, primer 1350 comprises a sequence complementary to a target sequence in an RNA molecule. In some cases, primer 1350 comprises a sequence that is complementary to a region of an immune molecule (such as a constant region of a TCR or BCR sequence). Primer 1350 hybridizes to nucleic acid molecule 1360, resulting in complementary molecule 1370. For example, the complementary molecule 1370 may be a cDNA generated in a reverse transcription reaction. In some cases, additional sequences may be added to the complementary molecule 1370. For example, reverse transcriptase may be selected such that several non-template bases 1380 (e.g., poly-C sequences) are added to the cDNA. In another example, terminal transferases may also be used to supplement the additional sequence. Nucleic acid barcode molecule 1390 comprises a sequence 1324 complementary to a non-template base, and reverse transcriptase performs a template switching reaction on nucleic acid barcode molecule 1390 to produce a barcoded nucleic acid molecule comprising a cell/cell nucleus (e.g., partition specific) barcode sequence 1322 (or reverse complement thereof) and a complementary molecule 1370 sequence (or portion thereof). In some cases, sequence 1323 comprises a sequence that is complementary to a region of an immune molecule (such as a constant region of a TCR or BCR sequence). Sequence 1323 hybridizes to nucleic acid molecule 1360 to produce complementary molecule 1370. For example, the complementary molecule 1370 may be produced in a reverse transcription reaction that produces a barcoded nucleic acid molecule comprising a cell/cell nucleus (e.g., partition specific) barcode sequence 1322 (or its reverse complement) and a complementary molecule 1370 sequence (or a portion thereof). Additional methods and compositions suitable for barcoding cdnas generated from mRNA transcripts (including those encoding V (D) J regions of immune cell receptors) and/or barcoding methods and compositions comprising template switching oligonucleotides are described in the following patents: international patent application WO2018/075693, U.S. patent publication No. 2018/0105808, U.S. patent publication No. 2015/0376609 filed on 6 months at 26, and U.S. patent publication No. 2019/0367969, each of which is incorporated herein by reference in its entirety for all purposes.

Single cell/single cell nuclear ATAC sequencing

The sample preparation methods, compositions, systems and kits disclosed herein can be used to process a variety of different types of nucleic acid molecules from single cells or single cell nuclei in units of individuals or in tandem, e.g., single cells or single cell nuclear suspensions derived from different sample types including liquid and solid tissue samples. The methods provided herein can allow for analysis of various deoxyribonucleic acid (DNA) molecules and/or ribonucleic acid (RNA) molecules from the same biological particle (e.g., a cell or cell component such as a cell nucleus). The DNA molecules and/or RNA molecules may be derived from biological particles. The analysis of different types of nucleic acid molecules may be performed simultaneously or near simultaneously. The methods provided herein can include the use of a partitioning scheme in which a material (e.g., different types of target nucleic acid molecules, such as target nucleic acid molecules contained within a cell or nucleus) is distributed between a plurality of partitions (such as a plurality of droplets or wells). The material (e.g., target nucleic acid molecules) may be first co-partitioned with one or more reagents to generate a barcoded nucleic acid product (e.g., within one of a plurality of partitions) corresponding to each of a variety of different target nucleic acid molecules (e.g., DNA molecules and RNA molecules), and then subjecting these barcoded nucleic acid products to one or more amplification processes (e.g., polymerase Chain Reaction (PCR), which may optionally be performed in bulk).

Biological particles (e.g., cells or nuclei) can be pre-sorted based on the transduced markers prior to analysis (e.g., as described herein) by Fluorescence Activated Cell Sorting (FACS) or Magnetic Activated Cell Sorting (MACS). For example, a fluorescent protein (e.g., GFP, YFP, CFP, mCherry, mRuby, etc.) may be used to transduce the cells.

Thus, the methods provided herein provide sample preparation techniques that allow for sequencing of nucleic acid molecules from single cells and single nuclei of interest. In eukaryotic genomes, chromosomal DNA winds itself around histone proteins (i.e. "nucleosomes") forming complexes called chromatin. Tight or loose packing of chromatin helps control gene expression. Tightly packed chromatin ("closed chromatin") typically does not allow gene expression, while more loosely packed accessible chromatin regions ("open chromatin") are associated with active transcription of the gene product. Methods for detecting the accessibility of whole genomic DNA have proven extremely effective in identifying regulatory elements across multiple cell types and quantifying changes that cause activation and/or inhibition of gene expression. One such method is to determine transposase accessible chromatin using high throughput sequencing (ATAC sequencing). The ATAC sequencing method detects DNA accessibility with an artificial transposon that inserts specific sequences into the accessible region of chromatin. Since transposases can insert sequences only into accessible regions of chromatin that are not bound by transcription factors and/or nucleosomes, sequencing reads can be used to infer regions of increased chromatin accessibility. Single cell ATAC-seq (scATAC-seq) methods have been developed as described in PCT patent publication No. WO2018/218226, U.S. patent nos. 10,844,372 and 10,725,027, and U.S. patent publication No. 20200291454, each of which is incorporated herein by reference in its entirety.

The methods, compositions, systems, and kits of the present disclosure can be used to prepare samples for downstream analysis using high throughput sequencing transposase accessible chromatin assays (ATAC-seq) and RNA sequencing (RNA-seq) assays, alone or in tandem. Such analysis may reveal regulatory factors that control cis-element accessibility and/or trans-factor occupancy. In some cases, the methods, systems, and kits provided herein can reveal nucleosome localization in a cell type and/or regulatory nodules that coordinate activity, such as coordinating trans factor activity, co-binding Transcription Factor (TF) to cis-element synergism, and the like. The methods provided herein can be performed in a high throughput manner in order to obtain single biological particle (e.g., single cell or single cell nucleus) data, including epigenomic variability.

In some embodiments, the biological particle is a cell or a nucleus. In some embodiments, the cell or nucleus is permeabilized. In some embodiments, the method further comprises lysing or permeabilizing the biological particles within the partition to provide access to the DNA molecules and/or RNA molecules therein. In some embodiments, the method further comprises treating the open chromatin structure of the biological particle with a transposase to produce a DNA molecule.

The nucleic acid molecule may undergo one or more processing steps within a biological particle (e.g., a cell or nucleus). For example, chromatin within a cell or nucleus may be contacted with a transposase. Examples of processing steps including transposon complexes can be found, for example, in U.S. patent publication 20180340171 and U.S. patent publication 20200291454, each of which is incorporated herein by reference in its entirety.

In one aspect, the present disclosure provides sample preparation techniques suitable for use in methods of processing DNA and/or RNA nucleic acid molecules from cells or nuclei. The method may comprise contacting the cell or cell nucleus with a transposase-nucleic acid complex comprising a transposase molecule and one or more transposon end oligonucleotide molecules. The cell or nucleus may be contacted with the transposase-nucleic acid complex in bulk solution such that the cell or nucleus undergoes "tag fragmentation" via a tag fragmentation reaction. Contacting the cell or nucleus with the transposase-nucleic acid complex can generate one or more template nucleic acid fragments (e.g., a "tag fragment"). The one or more template nucleic acid fragments may correspond to one or more target nucleic acid molecules (e.g., deoxyribonucleic acid (DNA) molecules) within a cell or nucleus. In parallel, the cell or cell nucleus may be contacted with a primer molecule (e.g., a primer molecule comprising a poly-T sequence) configured to interact with one or more additional target nucleic acid molecules (e.g., ribonucleic acid (RNA) molecules, such as messenger RNA (mRNA) molecules). The cells or nuclei may be contacted with the primer molecules in bulk solution. Alternatively, or in addition, the cells or nuclei may be contacted with primer molecules within the partition. Interactions between these moieties may result in one or more additional template nucleic acid fragments (e.g., RNA fragments). For example, a primer molecule may have at least partial sequence complementarity to one or more additional target nucleic acid molecules (e.g., mRNA molecules). The primer molecule may hybridize to the sequence of one additional nucleic acid molecule of the one or more additional nucleic acid molecules of interest. The cell or nucleus may be partitioned (e.g., co-partitioned with one or more agents) into one partition (e.g., of multiple partitions). For example, the partition may be a droplet or a hole. The partitions may comprise one or more reagents, including, for example, one or more particles (e.g., beads) comprising one or more nucleic acid barcode molecules. The cells or nuclei can be lysed, permeabilized, immobilized, crosslinked, or otherwise manipulated to provide an opportunity to access one or more template nucleic acid fragments and one or more additional template nucleic acid fragments therein. One or more template nucleic acid fragments and one or more additional template nucleic acid fragments therein may undergo one or more processing steps within the partition. For example, one or more template nucleic acid fragments and/or one or more additional template nucleic acid fragments may undergo a barcoding process, ligation process, reverse transcription process, template switching process, linear amplification process, and/or gap filling process. The resulting one or more treated template nucleic acid fragments (e.g., tag fragmentation fragments) and/or one or more treated additional template nucleic acid fragments (e.g., RNA fragments) can each include a barcode sequence (e.g., a nucleic acid barcode sequence, as described herein). One or more of the treated template nucleic acid fragments and/or one or more of the treated additional template nucleic acid fragments may be released from the partition (e.g., combined with the contents of other partitions of the plurality of partitions) and then may undergo one or more additional treatment steps in bulk. For example, one or more of the treated template nucleic acid fragments and/or one or more of the treated additional template nucleic acid fragments may undergo a gap filling process, a dA tailing process, a terminal transferase process, a phosphorylation process, a ligation process, a nucleic acid amplification process, or a combination of these processes. For example, one or more of the treated template nucleic acid fragments and/or one or more of the treated additional template nucleic acid fragments may be subjected to the following conditions: these conditions are sufficient to undergo one or more polymerase chain reactions (PCR, such as sequence independent PCR) to generate amplification products corresponding to one or more treated template nucleic acid fragments (e.g., tag-fragmented fragments) and/or one or more treated additional template nucleic acid fragments (e.g., RNA fragments). The sequences of such amplification products can be detected using, for example, a nucleic acid sequencing assay, and then used to identify the sequences of one or more target nucleic acid molecules (e.g., DNA molecules) and one or more additional target nucleic acid molecules (e.g., RNA molecules) of their source cells or nuclei. Additional methods, compositions, systems, and kits for processing DNA and/or RNA nucleic acid molecules from cells or nuclei are disclosed in U.S. patent publication No. 20200291454A1, which is incorporated herein by reference in its entirety.

One of the one or more nucleic acid barcode molecules may comprise a flow cell adaptor sequence, a barcode sequence, and a sequencing primer or portion thereof, which may be configured to interact (e.g., anneal or hybridize) with a complementary sequence contained in a template nucleic acid fragment of a DNA molecule derived from a biological particle (e.g., a cell or a cell nucleus) or derivative thereof (e.g., an ATAC sequencing fragment generated using a composition such as that of fig. 17-19).

FIG. 17 includes one example of a transposase-nucleic acid complex for use in the methods provided herein. Transposase-nucleic acid complex 1700 (e.g., comprising a transposase dimer) comprises partially double stranded oligonucleotide 1701 and partially double stranded oligonucleotide 1705. The partially double-stranded oligonucleotide 1701 comprises a transposon end sequence 1703, a first primer sequence 1702, and a sequence 1704 that is complementary to the transposon end sequence 1703. The partially double-stranded oligonucleotide 1705 comprises a transposon end sequence 1706, a first primer sequence 1707, and a sequence 1708 complementary to the transposon end sequence 1706. Primer sequences 1702 and 1707 may be the same or different. In some cases, primer sequence 1702 may be designated as a first sequencing primer, such as Illumina "R1" sequence, and primer sequence 1707 may be designated as a second sequencing primer, such as Illumina "R2" sequence. Transposon end sequences 1703 and 1706 may be the same or different.

FIG. 18 includes another example of a transposase-nucleic acid complex for use in the methods provided herein. Transposase-nucleic acid complex 1800 (e.g., including a transposable dimer) includes forked adaptors 1801 and 1806, which are partially double-stranded oligonucleotides. The partially double-stranded oligonucleotide 1801 comprises a transposon end sequence 1803, a first primer sequence 1802, a second primer sequence 1805, and a sequence 1804 complementary to the transposon end sequence 1803. The partially double-stranded oligonucleotide 1806 comprises a transposon end sequence 1807, a first primer sequence 1808, a second primer sequence 1818, and a sequence 1809 complementary to the transposon end sequence 1807. Primer sequences 1802, 1805, 1808, and 1810 may be the same or different. In some cases, primer sequences 1802 and 1808 may be designated as first sequencing primers, such as Illumina "R1" sequences, and primer sequences 1805 and 1810 may be designated as second sequencing primers, such as Illumina "R2" sequences. Alternatively, primer sequences 1802 and 1810 may be designated as "R1", and primer sequences 1805 and 1808 may be designated as "R2". Alternatively, primer sequences 1802 and 1808 may be designated as "R2", and primer sequences 1805 and 1810 may be designated as "R1". Alternatively, primer sequences 1802 and 1810 may be designated as "R2", and primer sequences 1805 and 1808 may be designated as "R1". Transposon end sequences 1803 and 1807 may be identical or different.

FIG. 19 shows a transposase-nucleic acid complex 1900 (e.g., comprising a transposase dimer) comprising hairpin molecules 1901 and 1906. Hairpin molecule 1901 comprises transposon end sequence 1903, first hairpin sequence 1902, second hairpin sequence 1905, and sequence 1904 complementary to transposon end sequence 1903. Hairpin molecules 1906 comprise transposon end sequences 1907, third hairpin sequences 1908, fourth hairpin sequences 1910, and sequences 1909 that are complementary to transposon end sequences 1907. Hairpin sequences 1902, 1905, 1908, and 1910 may be the same or different. For example, hairpin sequence 1905 can be the same as or different from hairpin sequence 1910, and/or hairpin sequence 1902 can be the same as or different from hairpin sequence 1908. Hairpin sequences 1902 and 1908 can be spacer sequences or adaptor sequences. Hairpin sequences 1905 and 1910 can be promoter sequences, such as T7 recognition or promoter sequences and/or UMI sequences. Transposon end sequences 1903 and 1907 may be the same or different. In some cases, sequence 1904 is a transposon end sequence, and sequence 1903 is a sequence complementary to sequence 1904. In some cases, sequence 1909 is a transposon end sequence, and sequence 1907 is a sequence complementary to sequence 1109.

Contacting a biological particle (e.g., a cell or nucleus) comprising one or more target nucleic acid molecules (e.g., DNA molecules) with a transposase-nucleic acid complex can generate one or more tagged (see, e.g., fig. 17-19) template nucleic acid fragments (e.g., "tag fragmentation fragments"). The one or more template nucleic acid fragments may each comprise the sequence of one or more target nucleic acid molecules (e.g., target sequences). The transposase-nucleic acid complexes can be configured to target a specific region of one or more target nucleic acid molecules (e.g., conjugated to an antibody specific for a protein that binds to a target sequence) to provide one or more template nucleic acid fragments comprising the specific target sequence. One or more of the template nucleic acid fragments may comprise a sequence of interest corresponding to accessible chromatin. A template nucleic acid fragment (e.g., a tag fragment) may comprise one or more gaps (e.g., between a transposon end sequence or its complement and a target sequence on one or both strands of a double stranded fragment). The gap can be filled via a gap filling process using an enzyme, such as a polymerase (e.g., a DNA polymerase). In some cases, an enzyme mixture may be used to repair the partially double stranded nucleic acid molecule and fill one or more gaps. Gap filling may not include strand displacement. Gaps inside or outside the partition may be filled.

Fig. 20 includes a general workflow of the methods provided herein. The left hand side of the figure includes chromatin workflow 2000. Tag fragmentation can be performed in batches to generate tag fragmented fragments of genomic DNA. The gDNA fragment can include one or more gaps (e.g., as described herein). The biological particles comprising one or more gDNA fragments can then be separated within a partition (e.g., a droplet or well) along with one or more reagents, wherein the reagents comprise a first nucleic acid barcode molecule and a second nucleic acid barcode molecule (e.g., as described herein). The one or more gDNA fragments of the partitioned biological particle and the first nucleic acid barcode molecule can be used to generate a first barcoded nucleic acid molecule. Gap filling may optionally be performed within the partitions before or after the first barcoded nucleic acid molecules are generated. In some cases, gap filling may be performed prior to partitioning. In other cases, gap filling may be performed after the first barcoded nucleic acid molecule or derivative thereof is recovered from the partition. After recovery from the partition, the first barcoded nucleic acid molecules or derivatives thereof may be subjected to additional processing, for example, to incorporate additional functional groups, including sequencing primers, flow cell adaptors, identification sequences, and the like, and/or to amplify such barcoded nucleic acid molecules or derivatives thereof to enrich the sample population for subsequent analysis, such as nucleic acid sequencing. The right hand picture of fig. 20 includes an RNA workflow 2050. After separation, an RNA molecule (e.g., an mRNA molecule) and a second nucleic acid barcode molecule, which may have the same or different sequence as the first nucleic acid barcode molecule, may be used to generate a second barcoded nucleic acid molecule. The second barcoded nucleic acid molecule or derivative thereof may be recovered from the partition along with the first barcoded nucleic acid molecule and then optionally subjected to additional treatment, for example, to incorporate additional functional groups including sequencing primers, flow cell adaptors, identification sequences, and the like, and/or to amplify such barcoded nucleic acid molecules or derivatives thereof to enrich the sample population for subsequent analysis, such as nucleic acid sequencing. The first and second barcoded nucleic acid molecules or their derivatives may be separated from each other prior to undergoing such additional treatment, or may be treated in a common (e.g., bulk) solution. Similarly, the first and second barcoded nucleic acid molecules or their derivatives or their amplicons may be subjected to nucleic acid sequencing at the same time and/or using the same system, or may be subjected to nucleic acid sequencing at different times and/or using different systems.

In one aspect, the sample preparation methods, kits, compositions, and systems described herein can be used as part of a nucleic acid analysis method, such as single cell/single cell nuclear based genomic DNA (gDNA) and optionally tandem RNA. Analysis of gDNA and RNA (e.g., mRNA) can be from the same cell or the same nucleus, and optionally can be performed simultaneously.

In one embodiment, the method includes the step of generating tissue fragments from a biological tissue sample. In another embodiment, the biological tissue sample is a solid tissue sample. In a further embodiment, the tissue fragments are substantially free of dissociated cells. In another embodiment, the method further comprises treating the tissue fragments with a fixation reagent to provide fixed tissue fragments. In other embodiments, the method further comprises dissociating the fixed tissue fragment to provide dissociated fixed tissue fragments comprising the fixed nuclei and/or fixed cells. In another embodiment, the dissociating step comprises treating the fixed tissue fragment with a cell dissociating agent and/or a unfixed agent. In one embodiment, the method further comprises generating a plurality of template nucleic acid fragments from and/or in the immobilized cell nucleus or immobilized cell using a plurality of transposase-nucleic acid complexes, wherein each transposase-nucleic acid complex comprises a transposase molecule and a transposon end oligonucleotide molecule. In another embodiment, the method further comprises generating a plurality of partitions. In some embodiments, the plurality of partitions are a plurality of droplets in an emulsion, or a plurality of pores. In another embodiment, one of the plurality of partitions includes: (i) A single fixed cell nucleus or a single fixed cell comprising one of the plurality of template nucleic acid fragments, and (ii) a single support having a plurality of barcode oligonucleotide molecules attached thereto, each barcode oligonucleotide molecule comprising a barcode sequence. In some other embodiments, the support is a particle, such as a bead, for example a gel bead. In a further embodiment, the method further comprises generating a barcoded nucleic acid fragment in the partition using at least (i) the template nucleic acid fragment and (ii) one of the plurality of barcode oligonucleotide molecules.

In another aspect, the fixed nuclei or fixed cells from the dissociated fixed tissue fragments undergo a step of de-fixation. In one embodiment, the step of unfixing comprises treating the fixed nucleus or fixed cell with a composition comprising an unfixing agent, thereby providing the unfixed nucleus or unfixed cell. In other embodiments, the composition comprises a detackifier selected from the group consisting of: compound (1), compound (2), compound (3), compound (4), compound (5), compound (6), compound (7), compound (8), compound (9), compound (10), compound (11), compound (12), compound (13), compound (14), compound (15), or a combination thereof. In another embodiment, the unfixed agent comprises compound (8), or both compound (1) and compound (8). In another embodiment, the composition may further comprise a protease. In one embodiment, the step of deammobilizing (to generate a deammobilized nucleus or a deammobilized cell) is performed prior to the step of generating a plurality of template nucleic acid fragments by using a plurality of transposase-nucleic acid complexes, wherein each transposase-nucleic acid complex comprises a transposase molecule and a transposon end oligonucleotide molecule. The unfixed step allows the generation of template nucleic acid fragments from or in the unfixed nuclei or unfixed cells.

H. Shredding device

The free hand slicing or chopping of tissue with a hand-held razor blade or other cutting device is a common technique for preparing biological samples from tissue. Pre-scoring dies have also been used to help chop tissue and provide accuracy compared to freehand methods. The mold may allow a user to make consistent coronal or sagittal cuts with the razor blade. Sliding microtomes are also available, but require training and practice, and also require careful temperature control during use (see, e.g., U.S. patent No. 7,827,894 and U.S. patent No. 1,865,539, each of which is incorporated herein by reference in its entirety). Vibrating microtomes and cryostat systems are also known, but may be expensive and/or require training to use (see, e.g., U.S. patent nos. 7,237,392 and 4,752,347, and U.S. patent publication No. 20160084741, each of which is incorporated herein by reference in its entirety).

Although the morcellation-fixation methods as disclosed herein may be implemented by morcellating tissue with a hand-held razor blade, the present disclosure also provides a mechanical morcellation device that may be used to morcellate tissue in a morcellation-fixation method. Generally, such a morcellating device includes a plurality of aligned razor blades configured with a mechanism for reproducibly holding a tissue sample within a morcellating disc, and assembled and disassembled by a user for cleaning.

Accordingly, in at least one embodiment, the present disclosure provides a shredding device comprising:

(a) A razor cartridge, preferably a circular cartridge, comprising a plurality of razor blade slots, each slot configured to insert a blade edge of a razor blade through the razor blade slot and to retain a top portion of the razor blade within the razor blade slot, wherein a bottom portion of the razor blade comprising the razor blade edge extends vertically below the razor cartridge;

(b) A razor blade alignment layer, preferably a circular layer, comprising a plurality of alignment openings, wherein each alignment opening is configured to insert a blade edge of a razor blade through the alignment layer such that a bottom portion of the razor blade comprising the razor blade edge extends below the alignment layer; and

(c) A sample tray holder configured to hold a sample tray containing a biological tissue sample;

Wherein the razor blade alignment layer is configured to be placed over and reversibly and rotatably connected to the sample disk holder such that each razor blade cutting edge extends into and toward the bottom of the sample disk holder.

Fig. 21 provides an unassembled exploded view of such a shredding device 100 useful in various shredding-securing methods of the present disclosure. The shredding device 2100 includes a razor cartridge 2110 aligned over a razor blade alignment layer 2120 which in turn is aligned over a sample tray 2130 which in turn is aligned over a sample tray holder 2140. Each of the components 2110, 2120, 2130 and 2140 are configured to be stacked as indicated in fig. 21 (and assembled as shown in fig. 23A).

Fig. 22 shows a top view of the assembled chopping device of fig. 21, including a razor blade alignment layer 2120 having a diameter greater than that of the razor cartridge 2110 and a centrally shown grip 2155, and screws 2154 on each side of the grip 2155.

Preferably, razor cartridge 2110 and razor blade alignment layer 2120 are configured to rotate relative to sample tray 2130 and/or tray holder 2140. Preferably, the razor cartridge, razor blade alignment layer, sample tray holder and sample tray are circular, as shown in fig. 21. In one embodiment, each of the components 2110, 2120, 2130 and 2140 is preferably circular as shown in fig. 21, and is preferably configured to fit together (aligned when stacked, as shown in fig. 23A).

The razor cartridge 2110 is preferably plate-like or disc-like as shown, having top and bottom surfaces and a circular side perimeter. The razor cartridge 2110 includes a plurality of razor blade slots/openings 2111 as shown in fig. 21, each slot/opening 2111 being configured for insertion of a razor blade cutting edge as shown.

As shown in fig. 21, the system preferably includes a razor blade 160 aligned over a plurality of razor blade slots/openings 2111. Each elongated razor blade slot/opening 2111 is configured to allow insertion of a single razor blade 2160. Preferably, the width of each slot/opening is large enough for blade 162 to be inserted, but small enough to prevent the entire razor blade from passing through. For example, it is preferred to use a single-edge razor blade 2160 having a back 2161, and if used, the width of the slot/opening 2111 is greater than the thickness of the blade 2162 (e.g., 0.010 inch slot width versus 0.009 inch blade thickness), but less than the thickness of the razor back 2161 (e.g., 0.012 inch back thickness), allowing the blade edge to be inserted through the opening, but the top of the razor blade to be held by the razor cartridge 2110.

Preferably, the length of each slot/opening ranges from 1 inch to 2 inches, more preferably from 1.3 inches to 1.7 inches, even more preferably from 1.4 inches to 1.6 inches.

Preferably, the length of each slot/opening is less than 0.5 inches longer than the length of the blade used, more preferably less than 0.2 inches longer than the length of the blade used, and even more preferably less than 0.1 inches longer than the length of the blade used.

Preferably, the width of each groove/opening ranges from 0.005 inch to 0.02 inch, more preferably from 0.006 inch to 0.014 inch, even more preferably from 0.009 inch to 0.0012 inch.

Preferably, the width of each slot/opening is less than 0.005 inches greater than the thickness of the blade edge of the blade used, more preferably less than 0.001 inches greater than the thickness of the blade edge of the blade used, and even more preferably less than 0.0005 inches greater than the thickness of the blade edge of the blade used.

As shown in fig. 21, alignment layer 2120 includes a plurality of alignment openings 2121 configured to align with razor blade slots/openings 2111, each slot/opening 2111 configured for insertion of a razor blade cutting edge and vertically aligning and stabilizing a razor blade. According to a preferred embodiment, the razor cartridge 2110 is circular, comprising at least two through holes 2113 and at least two screws 2114 configured to be inserted through the at least two through holes for reversibly connecting the circular razor cartridge 2110 to the razor blade alignment layer 2120. For example, the term "circular" as used in "circular razor cartridge" refers to the razor cartridge component being "circular" in contrast to defining the razor blades as circular. Preferably, each of the at least two screws 2114 is a spring-loaded screw (e.g., as shown in fig. 21, the shank and/or threads/length of the screw 2114 are surrounded by a spring 115) configured to provide a vertical lifting force to the circular razor cartridge relative to the alignment layer so as to provide a collapsible gap (shown as gap 2319 in fig. 23A) between the razor cartridge and the alignment layer. Preferably, spring 2115 is a compression spring.

According to a preferred embodiment, the circular razor cartridge comprises at least four through holes and at least four screws inserted through the at least four through holes for connecting the circular razor cartridge to the razor blade alignment layer.

Preferably, each of the at least four screws is a spring-loaded screw configured to provide a vertical lifting force to the circular shaving cartridge relative to the alignment layer so as to provide a collapsible gap between the razor cartridge and the alignment layer. The alignment layer 2120 preferably includes one or more depressions 2123 configured to receive the ends of the screws/springs 2114/2115, more preferably two or more depressions 2123, and even more preferably four or more depressions 2123. Fig. 23A is a side view showing the chopping device 2300 in a resting position (or normal or uncompressed position). The shredding device 2300 includes a handle 2350, a razor cartridge 2310, razor blades 2360, spring screws 2314/2315, an alignment layer 2320 and a sample tray holder 2340 as shown. The handle 2350 includes a grip 2355 connected to a base 2356 that is connected, preferably reversibly connected, to the top of a razor cartridge 2310. The shaving cartridge 2310 retains a plurality of shaving blades 2360 in a shaving cartridge opening (not shown). Alignment layer 2320 is shown mounted over sample disk holder 2340 and vertically aligns a plurality of razor blades 2360. According to an alternative embodiment, sample disk holder 2340 is omitted, and alignment layer 2320 is configured to reversibly fit over the open sample disk (not shown in fig. 23A).

Spring screws 2314/2315 connect the razor cartridge 2310 over the razor blade alignment layer 2320, wherein the springs 2315 are configured and/or biased to hold the razor cartridge 2310 over the alignment layer 2320, creating a compressible gap 2319 as shown in fig. 23A. Preferably, the spring 2315 is a compression spring. To actuate the cutting or slicing action, the handle 2350 is pressed downward to push the razor blade 2360 into a tissue sample held within a sample tray (not shown). Fig. 23B shows the razor cartridge 2310 being pushed down onto the alignment layer 2320, reducing or eliminating the gap 2319 as shown.

Fig. 23C is an elevational side view of the shredder of fig. 23A in a rest or normal position. Fig. 23D is an elevational side view of the shredding device of fig. 23A in a slicing or compressed position with the grip 2355 indicating an orientation in which the razor blades 2360 have been rotated relative to the sample tray holder 2340.

Preferably, the device is configured such that the blade edge of the razor blade is fully retracted into the aligned openings in the rest or uncompressed position, while material adhered to the blade edge is pushed away from the blade edge upon retraction.

Preferably, the device is configured such that the blade edge of the razor blade is retracted or lifted from the tissue or material in the sample tray so that the razor blade can be rotated relative to the sample tray without moving the tissue or material.

Preferably, the spring-loaded screw is configured such that the gap between the razor cartridge bottom and the alignment layer top is in the range of 0.1 inch to 0.5 inch (fig. 23A), more preferably in the range of 0.13 inch to 0.3 inch, and most preferably in the range of 0.15 inch to 0.2 inch in the rest or uncompressed position.

According to an alternative embodiment, the shredding device comprises at least two springs, preferably at least four springs, configured to be placed between the circular razor cartridge and the alignment layer. According to a preferred embodiment, the shredding means comprises a spring, preferably a compression spring, surrounding each screw.

According to an alternative embodiment, the shredding means comprises springs (not shown in fig. 21) separate from the screws, more preferably the bottom surface of the razor cartridge and/or the top surface of the alignment layer comprises recesses (not shown in fig. 21) configured to receive the ends of each spring to fix the position and/or orientation of the springs. According to a preferred embodiment, the shredding device further comprises at least four springs configured to be placed between the circular razor cartridge and the alignment layer.

According to a preferred embodiment, the device includes two or more screws, rivets, bolts or other means for connecting the razor cartridge 2310 to the razor blade alignment layer 2320, and two or more springs, preferably two or more compression springs, located between the razor cartridge 2310 and the razor blade alignment layer 2320. Further, preferably, the one or more springs encircle one or more screws, rivets, bolts or other connection means.

According to a preferred embodiment, as shown in FIG. 21, the shredding device includes a handle 2150 connected to the top of the circular razor cartridge 2110. Preferably, the handle 2150 is connected to the circular razor cartridge with at least one screw 2154, more preferably with at least two screws 2154. Preferably, the handle 2150 includes one or more through holes 2153 for inserting one or more screws 2154.

Preferably, the handle 2150 includes a grip 2155 that is configured to facilitate assembly and/or use of the shredder 2100. For example, grip 2155 is configured to allow handle 2150 to rotate.

According to another preferred embodiment, the handle comprises a disc having at least one through hole for a screw to connect the handle to the circular razor cartridge. Preferably, the top side of the disc includes a handle or grip member.

Preferably, handle 2150 includes grip 2155 connected to handle base 2156. According to a preferred embodiment, grip 2155 and handle base 2156 are integral or single components. According to alternative embodiments, grip 2155 is attached to handle base 2156 (via glue, screws, interlocking mechanisms, or other connection means).

According to a preferred embodiment, the handle 2150 includes one or more through holes 153 for inserting one or more screws 2154 through the handle base 2156.

According to a preferred alternative embodiment, the circular razor cartridge includes a top handle or grip, such as a handle or grip integral with the circular razor cartridge, but configured to allow insertion of the razor blades 2160 into the razor blade slots/openings 2111 (not shown).

According to a preferred alternative embodiment, the handle is configured to reversibly snap or screw onto the top of the circular razor cartridge.

Preferably, the handle is configured to retain the razor blade in the circular shaving cartridge (e.g., reversibly lock the razor blade into the slot opening) after insertion of the razor blade into the circular shaving cartridge and the circular shaving blade alignment layer.

According to an alternative embodiment, the device comprises a dome-shaped razor cartridge having a slot/opening through a bottom surface from the top of the dome, and the dome is configured to provide a handle for use of the device and a means for holding the razor blade. According to one embodiment, the razor blades fall into these slots/openings and are reversibly locked into place (e.g., locking the top insert, locking mechanism within the dome, and a rod inserted through the opening in the dome and through hole 2169 in the blade to lock into place).

According to a preferred embodiment, the circular razor blade alignment layer 2120 has an alignment layer diameter and the plurality of alignment openings 2121 are parallel and have a length of at least 75% of the frame diameter.

According to a preferred embodiment, the circular razor cartridge 2120 has a cartridge diameter, and the plurality of razor blade slots are parallel and have a length of at least 85% of the cartridge diameter.

Preferably, the circular razor cartridge 2120 has a cartridge diameter and the circular razor alignment layer 2120 has an alignment layer diameter that is greater than the cartridge diameter.

According to a preferred embodiment, the circular razor blade alignment layer 2120 includes an annular raised surface 2127 about the outer perimeter of the top surface, thereby creating a circular recessed central surface 2128 having a recessed central diameter surrounded by the annular raised surface, wherein the recessed central diameter is sized to fit the diameter of the circular razor cartridge 2110. Preferably, the annular raised surface is 0.5mm to 10mm higher than the circular recessed central surface, more preferably 1mm to 4mm higher, most preferably about 2mm higher.

The device is preferably configured for use with conventional sample plates or sample trays.

The apparatus is preferably configured to be used as a hand-held device for slicing or homogenizing tissue samples.

According to a preferred embodiment, the device comprises at least one reusable sample plate or sample tray 2130.

According to a preferred embodiment, the device includes a circular sample tray holder 2140 configured to hold at least one reusable sample plate or sample tray 2130, as shown in FIG. 21. Preferably, circular sample disk holder 2140 is a bowl configured to hold sample disk 2130.

Preferably, the circular sample disk holder is a cylindrical disk configured to hold a cylindrical sample disk.

According to a preferred embodiment, the circular sample disk holder has a disk holder outer height, a disk holder inner height (e.g., disk holder outer wall 2141), a disk holder outer diameter, and a disk holder inner diameter. Preferably, the device further comprises a circular sample tray 2130 having a sample tray outer height 2131, a sample tray inner height, a sample tray outer diameter, and a sample tray inner diameter. Preferably, the disk holder inner diameter is larger than the sample disk outer diameter and the disk holder inner height is larger than the sample disk outer height.

Preferably, bottom surface 2143 of circular sample tray holder 2140 is configured to correspond to bottom surface 2133 of sample tray 2130.

Preferably, the circular razor cartridge and circular razor blade alignment layer are configured such that when the circular razor cartridge and circular razor blade alignment layer are pushed down or in a "slicing position" or "compression position", the blade edge of each razor blade inserted into the razor blade slot and opening will contact the bottom of the sample tray (see fig. 23B).

Fig. 24A is a side view of the sectioning device 2400 in a "rest position" indicated by an uncompressed gap 2419. Preferably, the height of the shredding device when in a rest or uncompressed position (e.g., FIG. 24A) ranges from 2 inches to 4 inches, preferably from 2 inches to 2.5 inches.

Fig. 24B is a side cross-sectional view of the device of fig. 24A in a compressed or slicing position, as shown, with each cutting edge 2462 of the razor blade 2460 contacting an interior bottom surface 2443 of the sample disk 2440 such that tissue (not shown) within the sample disk 2440 will be sliced by the cutting edge of the razor blade.

According to a preferred embodiment, circular sample disk holder 2440 includes a top circular edge 2448 around the circumference of the holder, and alignment layer 2420 includes an annular channel 2426 around the outer circumference of the circular bottom surface and corresponding to top circular edge 2448 of the sample disk holder, wherein annular channel 2426 is configured to receive top circular edge 2448 to reversibly and rotatably couple alignment layer 2420 onto the top of circular sample disk holder 2440.

Preferably, the alignment layer 2420 includes a second annular channel 2427 around the outer perimeter of the circular bottom surface and within the inner circumference of the annular channel 2426, wherein the second annular channel 2427 is configured to receive the top circular edge of the sample disk 2430 held within the circular sample disk holder 2440.

Fig. 25 is a side cross-sectional view of a shredder 2500 in a "resting mode" or "normal" position indicated by gap 2519 according to another embodiment. Razor blade cutting edge 2562 is shown above the bottom inner surface of sample disk 2530 and is nearly fully retracted into alignment layer 2520. Sample disk holder 2540 includes a top edge 2548, and the bottom side of alignment layer 2520 includes an annular channel 2526, which is configured to receive top edge 2548 as shown. The bottom side of alignment layer 2520 preferably also includes a second annular channel 2527 located within annular channel 2526, which is configured to receive edge 2538 of sample tray 2530 as shown. Annular channel 2526/2527 allows alignment layer 2520 to be rotatably attached over sample disk holder 2540 and sample disk 2530.

Preferably, the height of the circular razor cartridge 2510 ranges from 1mm to 20mm, more preferably from 2mm to 10mm, even more preferably from 4mm to 6mm, most preferably about 5mm.

Preferably, the thickness (or height) of the alignment layer 2520 ranges from 5mm to 30mm, more preferably from 10mm to 20mm, even more preferably from 10mm to 15mm, and most preferably about 13.5mm.

Preferably, the outer height of circular sample disk holder 2540 ranges from 5mm to 30mm, more preferably from 10mm to 25mm, even more preferably from 15mm to 20mm, most preferably about 16mm.

Preferably, the razor cartridge 2510 is circular with a diameter in the range of 1.5 inches to 2.5 inches, more preferably 1.75 inches to 2.25 inches, even more preferably 1.85 inches to 2 inches.

Preferably, the alignment layer 2520 is circular with an outer diameter ranging from 50mm to 100mm, more preferably from 60mm to 90mm, even more preferably from 65mm to 75mm, most preferably about 69mm.

Preferably, alignment layer 2520 has a circular inner diameter within the circular sample disc holder during use, which ranges from 50mm to 100mm, more preferably from 60mm to 90mm, even more preferably from 65mm to 75mm, most preferably about 51.75mm.

Preferably, sample disk holder 2540 is preferably circular with an inner diameter in the range of 40mm to 200mm, more preferably 40mm to 100mm, even more preferably 55mm to 75mm, and most preferably about 60mm.

Preferably, sample disk holder 2540 is preferably circular with an outer diameter in the range of 40mm to 200mm, more preferably 40mm to 100mm, even more preferably 55mm to 75mm, and most preferably about 64mm.

Fig. 26 shows a side view of a cutting device 2600 according to another embodiment of the invention, including a razor blade 2660, a sample disk 630, and a sample disk holder 2640. The cutting device 2600 includes a handle 2650, a razor cartridge 2610, and an alignment layer 2620. The handle 2650 includes a grip 2655 and a base 2656 and is configured to be reversibly coupled to the top of the razor cartridge 2610 using screws or bolts 2654.

Razor blade 2660 is preferably eleven (11) single-edge blades, each having a cutting edge 2662 and a back 2661. The razor cartridge 2610 includes a plurality of razor blade slots (not shown) for inserting the blade edges 2662, but are configured to be narrower than the backs 2661 to retain the razor blades 2660 within the razor cartridge 2610.

The razor cartridge 2610 is configured to be reversibly connected to the top of the alignment layer 2620 using two or more screws 2614 (or bolts). As shown, the screw 2614 is preferably a shoulder screw that includes a top handle portion 2617 configured to be threaded into a bottom handle portion 2618, the bottom handle portion 2618 configured to fit within a compression spring 2615. These shoulder screws are preferably used for screws 2654 and/or screws 2614 and/or other mechanical fasteners for providing a bottom portion of a different diameter than a top portion and/or allowing the bottom portion to freely rotate to provide a freely rotatable pin joint connection with another component (e.g., connecting the razor cartridge to an alignment layer).

The compression spring 2615 is configured to surround a bottom portion 2618 of the screw 2614 and is configured to provide a compressible gap between the razor cartridge 2610 and the alignment layer 2620. The alignment layer 2620 preferably includes two or more recesses (not shown) on the top surface for receiving the screws 2614 and the springs 2615. Alignment layer 2620 preferably includes a central recessed region on the top surface for receiving razor blade portion 2618 (as discussed above and below).

According to a preferred embodiment, alignment layer 2620 has a top portion 2622 and a bottom portion 2624 (or structure connected to the bottom of top portion 2622) configured to provide an opening 2625 for the blade 2662 of razor blade 2660, and also to provide one or more retaining members 2626 configured to retain a tissue sample (not shown) in the bottom of sample tray 2630 below blade 2662 of razor blade 2660 and to prevent the tissue sample (and/or reduce tissue) from being pushed to the sides of the bottom of sample tray 2630.

Preferably, the opening a (between the retaining members 2626) is configured and dimensioned to receive the blades of the plurality of blades (e.g., to allow the blades to pass downwardly into the volume of the sample tray to contact tissue received therein). That is, the blade, when surrounded by the retaining member 2626, is allowed to pass through the opening a and contact the bottom of the sample tray.

Preferably, the height B of the retaining member 2626 is configured such that when the alignment layer 2620 is attached to the sample tray 2630 (e.g., the shredder 2600 is fully assembled for use) and the shredder is compressed or in the slicing position, the bottom surface of the retaining member 2626 contacts the bottom surface of the sample tray 2630.

Preferably, retaining member 2626 is recessed from the outer circumference of top portion 2622, creating a bottom recessed area C configured to accommodate a first annular channel (not shown) for receiving the top edge of sample disk 2630 and a second annular channel (not shown) for receiving the top edge of sample disk holder 2640.

Fig. 27A-27D are schematic diagrams of an alignment layer 2700 according to another preferred embodiment of the present disclosure. The alignment layer 2700 is preferably circular and preferably has an outer diameter 2702 in the range: 65mm to 75mm, more preferably 68mm to 70mm, most preferably about 69mm, and a total fixed height in the range of: preferably 10mm to 15mm, more preferably 12mm to 14mm, most preferably about 13.5mm.

Fig. 27A is a top view of a circular alignment layer 2700 comprising an annular raised surface 2727 on a top surface around an outer circumference and a surrounding circular recessed central surface 2728 having a recessed central diameter 2704, wherein the recessed central diameter is sized to fit the diameter of a circular razor cartridge (not shown). Preferably, the recess center diameter 2704 ranges from 40mm to 60mm, more preferably from 45mm to 55mm, even more preferably from 48mm to 52mm, most preferably about 50mm.

Preferably, the annular raised surface 2727 is 0.5mm to 10mm higher than the circular recessed central surface 2728, more preferably 1mm to 2mm higher, and most preferably about 1.5mm higher.

As shown in fig. 27C, alignment layer 2700 has a top portion 27122 and a bottom portion 27124 (or structure connected to the bottom of top portion 27122), wherein bottom portion 27124 is configured to provide an opening 27125 for receiving a razor blade cutting edge, and bottom portion 27124 further provides one or more retaining members 27126 configured to retain a tissue sample (not shown) centrally under the razor blade in the bottom of the sample tray and to prevent the tissue sample (and/or reduce tissue) from being pushed to the sides of the bottom of the sample tray during a slicing action. Preferably, each retaining member 27126 has a retaining wall 27226 configured to retain tissue between the retaining members 27126 along with opposing retaining walls 27226 during a slicing action of the razor blade.

Preferably, the width 27128 of the opening 27125 (between the walls 27227 of the retaining member 27126) is configured and dimensioned to receive the cutting edges of a plurality of blades. That is, the blade, when surrounded by the retaining member 27126, is allowed to pass through the opening a and contact the bottom of the sample tray. Preferably, the width 27128 ranges from 20mm to 40mm, more preferably from 25mm to 35mm, even more preferably from 28mm to 29mm, and most preferably about 28.5mm.

Preferably, the retaining member height 27130 is configured such that when the alignment layer 2700 is attached to a sample tray (e.g., the shredding device is fully assembled for use) and the shredding device is compressed or in a slicing position, the bottom surface of the retaining member 27126 contacts the bottom surface of the sample tray. Preferably, the height 27130 ranges from 4mm to 20mm, more preferably from 6mm to 12mm, more preferably from 8mm to 9mm, and most preferably about 8.5mm.

Preferably, retaining member 27126 is recessed from the outer circumference of top portion 27122, creating a bottom recessed region 27135 configured to accommodate a first channel 2727 (shown in fig. 27B) for receiving a top edge (not shown) of a sample tray and a second annular channel 2726 (shown in fig. 27B) for receiving a top edge (not shown) of a sample tray holder. Preferably, the recessed region 27135 has a width in the range of 15mm to 20mm, more preferably 17mm to 18mm, and most preferably about 17.25mm.

Preferably, the width of the first annular channel 2727 ranges from 2mm to 2.5mm, more preferably from 2.1mm to 2.2mm, and most preferably is about 2.13mm. Preferably, the width of the second annular channel 2728 ranges from 2mm to 4mm, more preferably from 2.5mm to 3.5mm, and most preferably about 3mm.

Fig. 27D is a bottom view of alignment layer 2700, showing first and second annular channels 2727 and 2728, as well as retaining members 27126 and openings 27125, with a plurality of razor slots 2721 aligned within openings 27125 between retaining walls 27226, as shown.

Fig. 27A-27D illustrate various dimensions of an alignment layer assembly 2700, which illustrates a preferred embodiment of the present invention. Other preferred embodiments may include one or more different sizes having substantially the same approximate dimensions.

Fig. 28A-28B are illustrations of an alignment layer 2800 according to another preferred embodiment. Fig. 28A is a top perspective view of an alignment layer 2800 having a top surface comprising a recessed area 2828 including a plurality of aligned razor slots 2821 and four screw holes 2823, the alignment layer configured to be attached to a circular razor cartridge with screws (not shown). The recessed region 2828 is shown within the raised annular region 2827 and is configured to receive the bottom (not shown) of a circular shaving cartridge.

Fig. 28B is a bottom perspective view of the alignment layer 2800 showing the razor slot 2821 between the retaining members 28126 having walls 28226. Fig. 28B also shows a first annular channel 2827 positioned within the second annular channel 2826. Fig. 28B shows retaining walls 28226 of retaining member 28126 configured to enclose a tissue sample between retaining walls 28226 of two retaining members 28126 when the device is compressed to retain tissue under a razor blade.

The morcellating device is preferably configured to hold at least one razor blade, preferably a plurality of razor blades, more preferably at least two razor blades, even more preferably at least five razor blades, even more preferably at least ten razor blades, and most preferably eleven razor blades, configured to slice or homogenize tissue or a biological sample or specimen. According to a preferred embodiment, the device is configured to hold or comprise at least five razor blade slots and at least five aligned openings, more preferably at least ten razor blade slots and at least ten aligned openings, preferably less than twenty (20) blades. According to a preferred embodiment, the razor cartridge comprises at least eleven razor slot openings and the razor blade alignment layer comprises at least eleven alignment openings.

According to a preferred embodiment, the razor blade slot openings include a top slot opening configured to hold the top of a razor blade (e.g., a thicker blade back of a single-edge razor blade) and a bottom slot opening configured to allow the sharp edge of the blade to pass through. Preferably, each razor blade slot opening ranges from 35mm to 45mm in length and from 0.1mm to 0.4mm in width. Preferably, the top slot opening has a size in the range of 0.5mm to 3mm by 30mm to 50mm, more preferably 1.0mm to 2mm by 35mm to 45mm, and most preferably about 1.3mm by 40.64mm. Preferably, the bottom slot opening has a size in the range of 0.1mm to 1.5mm by 30mm to 50mm, more preferably 0.3mm to 0.8mm by 35mm to 45mm, and most preferably about 0.4mm by 40.4mm.

According to a preferred embodiment, the shredding device or kit comprises, or is configured to hold, a plurality of razor blades, wherein the cutting edge of each razor blade is configured to fit through each razor blade slot and each aligned opening. Preferably, each aligned opening has a length in the range of 30mm to 50mm, more preferably 35mm to 45mm, most preferably about 40.4mm, and a width in the range of 0.1mm to 1.0mm, more preferably 0.3mm to 0.5mm, most preferably about 0.4mm.

Preferably, each razor blade is a single-edge razor blade, preferably including a top blade back (shown in fig. 21 as blade back 2161).

Preferably, each razor blade has a length in the range of 35mm to 45mm and a thickness in the range of 0.1mm to 0.4mm. According to a preferred embodiment, each razor blade is about 1.5 inches in length, about 11/16 inches in width, and about 0.009 inches in thickness.

According to another embodiment, the shredding device or shredding device kit comprises a sample tray, preferably a circular sample tray. Preferably, the sample tray has an outer diameter in the range of 40mm to 65mm, more preferably 50mm to 60mm, most preferably 54mm to 55.25mm, a tray outer height in the range of 10mm to 20mm, more preferably 12mm to 15mm, most preferably about 13.93mm, a tray inner height in the range of 7mm to 20mm, more preferably 10mm to 15mm, most preferably about 12.43mm, an inner diameter in the range of 39mm to 59mm, more preferably 44mm to 55mm, most preferably about 52.75mm.

According to an alternative embodiment, the chopping device is used without a sample disc holder, the alignment layer being rotatably attached directly to the sample disc.

In another aspect, the present disclosure provides a tissue preparation kit comprising at least one morcellating device and/or one or more morcellating device components (e.g., razor cartridge, razor blade alignment layer, and sample tray holder) configured for assembly in one or more containers. In at least one embodiment of the invention directed to a kit comprising a shredding device as described herein and at least one blade, preferably at least one razor blade, more preferably a plurality of razor blades.

According to a preferred embodiment, the kit further comprises a plurality of razor blades, preferably at least ten razor blades. According to a preferred embodiment, the kit further comprises at least one sample tray or sample plate. According to a preferred embodiment, the kit further comprises at least one sample tray holder. According to a preferred embodiment, the kit further comprises two or more screws and/or springs, preferably compression springs. According to another preferred embodiment, the kit comprises two or more alignment layers of different dimensions (e.g. diameter, annular channel width) and/or two or more sample tray holders of different dimensions (e.g. diameter) to accommodate sample trays of different sizes and designs.

In another aspect, the present disclosure also provides a method of using a shredding device as described herein. In at least one embodiment of a method of using the disclosed chopping device, the method includes pressing down on the razor cartridge, thereby forcing the cutting edges of the plurality of razor blades against and cutting the tissue sample.

In at least one embodiment of a method of using a lancing device of the present disclosure, wherein the lancing device comprises a plurality of razor blades inserted into a plurality of razor blade slots and through a plurality of aligned openings, placed over and reversibly and rotatably connected to a circular sample disk holder containing an open circular sample disk containing a tissue sample, the method comprises:

(i) Pressing down on the razor cartridge, thereby forcing the cutting edges of the plurality of razor blades against and cutting the tissue sample;

In at least one embodiment of the method, the shredding device is configured such that each depression of the plurality of razor blades causes the plurality of razor blades to be offset at least 10 degrees relative to the orientation of the sample tray (e.g., external threads within the sample tray holder, and the alignment layer and spring cause automatic reorientation after each depression action).

In at least one embodiment, the method of using the morcellating device further comprises rotating the plurality of razor blades to a third orientation relative to the tissue sample, and then pressing the razor cartridge down onto the circular sample tray holder, thereby forcing the cutting edges of the plurality of razor blades to press onto the tissue sample in the second orientation and cut the tissue sample. Preferably, the plurality of razor blades are rotated at least 10 to 25 degrees, preferably 15 to 20 degrees, per rotation.

In at least one embodiment of the method of using the shredding device, the method further comprises, after cleaning the shredding device, re-using it to shred or homogenize another sample, preferably the method further comprises disassembling the shredding device and cleaning the disassembled parts, and reassembling the parts prior to the next use. In at least one embodiment, the method further comprises using the blade after the blade is replaced to chop or homogenize another sample.

Additional embodiments of the present disclosure are provided in the following numbered clauses.

1. A method for analyzing nucleic acid, comprising

a) Generating tissue fragments from a biological tissue sample;

b) Treating the tissue fragments with a fixation reagent to provide fixed tissue fragments;

c) Dissociating the fixed tissue fragments to provide dissociated fixed tissue fragments comprising fixed nuclei; and

d) Generating a plurality of template nucleic acid fragments in the immobilized cell nucleus using a plurality of transposase-nucleic acid complexes, each complex comprising a transposase molecule and a transposon end oligonucleotide molecule.

2. The method of clause 1, further comprising e) generating a plurality of partitions, wherein one partition of the plurality of partitions comprises: (i) A single immobilized cell nucleus comprising one of the plurality of template nucleic acid fragments, and (ii) a plurality of barcode oligonucleotide molecules each comprising one barcode sequence.

3. The method of clause 2, further comprising f) generating a barcoded nucleic acid fragment in the partition using at least (i) the template nucleic acid fragment and (ii) one of the plurality of barcode oligonucleotide molecules.

4. A method for analyzing nucleic acid, comprising

a) Generating tissue fragments from a biological tissue sample;

d) Contacting the open chromatin structure of an immobilized cell nucleus with a transposase-nucleic acid complex to produce the immobilized cell nucleus comprising a tagged fragmented fragment of a deoxyribonucleic acid (DNA) molecule, wherein the immobilized cell nucleus further comprises ribonucleic acid (RNA).

5. The method of clause 4, further comprising e) generating a plurality of partitions, wherein one partition of the plurality of partitions comprises (i) the fixed nucleus, (ii) a first nucleic acid barcode molecule comprising a first barcode sequence, (iii) a second nucleic acid barcode molecule comprising a second barcode sequence, and (iv) a splint molecule, wherein the first nucleic acid barcode molecule comprises an overhang sequence, and wherein the splint molecule comprises a first sequence complementary to the sequence of the tag fragment and a second sequence complementary to the overhang sequence.

6. The method of clause 5, further comprising, within the partition: (i) Generating a first barcoded nucleic acid product comprising the first barcode sequence or its reverse complement and the DNA molecule sequence using the tag fragmentation fragment, the first nucleic acid barcode molecule, and the splint molecule, and (ii) generating a second barcoded nucleic acid product comprising the second barcode sequence or its reverse complement and a complementary DNA (cDNA) sequence of the RNA molecule using the RNA molecule and the second nucleic acid barcode molecule.

7. A method for analyzing nucleic acid, comprising

a) Generating tissue fragments from a biological tissue sample;

c) Dissociating the fixed tissue fragments to provide dissociated fixed tissue fragments comprising fixed nuclei;

d) Debarking the immobilized cell nuclei with a debarking reagent; and

e) Generating a plurality of template nucleic acid fragments in the disarmed cell nucleus using a plurality of transposase-nucleic acid complexes, each complex comprising a transposase molecule and a transposon end oligonucleotide molecule.

8. The method of clause 7, further comprising e) generating a plurality of partitions, wherein one partition of the plurality of partitions comprises: (i) A single unfixed cell nucleus comprising one of the plurality of template nucleic acid fragments, and (ii) a plurality of barcode oligonucleotide molecules each comprising one barcode sequence.

9. The method of clause 8, further comprising f) generating a barcoded nucleic acid fragment in the partition using at least (i) the template nucleic acid fragment and (ii) one of the plurality of barcode oligonucleotide molecules.

10. A method for analyzing nucleic acid, comprising

a) Generating tissue fragments from a biological tissue sample;

d) Debarking the immobilized cell nuclei with a debarking reagent; and

e) Contacting the open chromatin structure of the deblocked cell nucleus with a transposase-nucleic acid complex to produce the deblocked cell nucleus comprising a tagged fragmented fragment of a deoxyribonucleic acid (DNA) molecule, wherein the deblocked cell nucleus further comprises ribonucleic acid (RNA).

11. The method of clause 10, further comprising e) generating a plurality of partitions, wherein one partition of the plurality of partitions comprises (i) the unfixed nucleus, (ii) a first nucleic acid barcode molecule comprising a first barcode sequence, (iii) a second nucleic acid barcode molecule comprising a second barcode sequence, and (iv) a splint molecule, wherein the first nucleic acid barcode molecule comprises an overhang sequence, and wherein the splint molecule comprises a first sequence complementary to the sequence of the tag-fragmented fragment, and a second sequence complementary to the overhang sequence.

12. The method of clause 11, further comprising, within the partition: (i) Generating a first barcoded nucleic acid product comprising the first barcode sequence or its reverse complement and the DNA molecule sequence using the tag fragmentation fragment, the first nucleic acid barcode molecule, and the splint molecule, and (ii) generating a second barcoded nucleic acid product comprising the second barcode sequence or its reverse complement and a complementary DNA (cDNA) sequence of the RNA molecule using the RNA molecule and the second nucleic acid barcode molecule.

13. The method of any one of preceding clauses 1 to 12, wherein the dissociating step comprises treating the fixed tissue fragment with a cell dissociating agent.

14. The method of any one of preceding clauses 1 to 13, wherein the tissue fragments from step a) are substantially free of dissociated cells.

15. The method of any one of preceding clauses 1 to 14, wherein the biological tissue sample is a solid tissue sample.

16. The method of any one of preceding clauses 1 to 15, wherein the plurality of barcode oligonucleotide molecules are attached to a support.

17. The method of clause 16, wherein the support is a bead.

18. The method of clause 17, wherein the bead is a gel bead.

19. The method of clause 16, wherein the plurality of barcode oligonucleotide molecules are releasably attached to the support.

20. The method of any one of preceding clauses 1 to 19, further comprising lysing or permeabilizing the fixed or unfixed nuclei to provide access to the tag-fragmented fragments.

21. The method of any one of preceding clauses 1 to 20, wherein the transposase-nucleic acid complex comprises a first adapter and a second adapter, and wherein the tag-fragmented fragment comprises sequences of the DNA molecule flanking the first adapter and the second adapter.

22. The method of clause 12, wherein (i) comprises hybridizing the splint molecule to: a) The first adaptor or the second adaptor of the tag-fragmented fragment, and B) the first nucleic acid barcode molecule, and then ligating the first nucleic acid barcode molecule and the tag-fragmented fragment to generate the first barcoded nucleic acid product.

23. The method of clause 22, wherein the first adapter comprises a first transposon end sequence and a first primer sequence, and wherein the second adapter comprises a second transposon end sequence and a second primer sequence.

24. The method of clause 23, wherein (i) comprises a) hybridizing the splint molecule to: 1) The first primer sequence or the second primer sequence of the tag-fragmented fragment, and 2) the first nucleic acid barcode molecule, and B) ligating the first nucleic acid barcode molecule and the tag-fragmented fragment to generate the first barcoded nucleic acid product.

25. The method of clause 22, wherein the first primer sequence or the second primer sequence is single stranded.

Additional methods, compositions, systems, and kits for processing DNA and/or RNA nucleic acid molecules from cells or nuclei are disclosed in U.S. patent publication No. 20200291454A1, which is incorporated herein by reference in its entirety.

Examples

Various features and embodiments of the present disclosure are shown in the following representative examples, which are intended to be illustrative and not limiting. Those skilled in the art will readily appreciate that the specific examples are merely illustrative of embodiments of the disclosure that are more fully described in the claims that follow. Each embodiment and feature described in this application should be understood to be interchangeable and combinable with each embodiment contained therein.

Example 1: preparation of mouse brain tissue as dissociated fixed cells by a chopper-fixed method

This example illustrates the preparation of dissociated PFA-immobilized cells from mouse brain tissue using a chopper-immobilized method. The dissociated PFA immobilized cell biological samples prepared by this method can be used in a series of partition-based single-cell assays, including RNA template ligation or reverse transcription of nucleic acids, as described in further examples below.

Materials and methods:

A. cutting up tissue

Fresh mouse brain tissue samples were placed in clean petri dishes on ice until ready to be minced. Brain tissue was minced by hand using a single clean razor blade, with the step size between each cut being as small as possible. Care is taken to avoid dragging or swirling the tissue during the chopping process. The shredding process was repeated on 3 different axes for the entire brain tissue to minimize fine shredding of the samples. The resulting minced mouse brain tissue consisted of minced tissue particles having an average size of 0.5mm x 0.5 mm.

B. Fixation of minced tissue particles

The finely minced particles of mouse brain tissue were immediately immersed in a 4% PFA solution. Control samples were also prepared with minced tissue, but were not immobilized by direct immersion of minced tissue particles in a dissociation reagent solution. The ratio of the volume of 4% PFA solution to the volume of tissue in the tube was 10 times and the tube was left overnight at 4 ℃. After overnight storage, the samples were centrifuged at 500rcf for 5 minutes. The supernatant of the fixation solution was removed. The fixed tissue at the bottom of the tube was resuspended with cell resuspension buffer (1% BSA+0.2U/mL RNase inhibitor PBS).

C. Dissociating the immobilized tissue particles

The resuspended sample was transferred to a new tube using a wide-mouth P1000 pipette and centrifuged again (at 500 rcf) for 2 minutes. The majority of the resuspension buffer was removed and the remaining approximately 1mL (as a slurry of fixed brain tissue (250 mg tissue)) was transferred to a 4mL tube containing 1mL of dissociation reagent, i.e., collagenase A (Roche KGaA, darmstadt, DE; millipore-Sigma catalog number 11088793001) at a concentration of 2.5mg/mL in PBS. The fixed tissue in the dissociating agent mixture was shaken at 700rpm for about 90 minutes at 37 ℃.

While still in the tube, the tissue dissociation reagent mixture was pipetted 20 times with a wide-mouth P1000 pipette and then 20 times with a plain-mouth P1000 pipette. After pipetting, the tubes were centrifuged again, washed and resuspended twice in 1% BSA in PBS+ribonuclease inhibitor (Roche KGaA, darmstadt, DE; millipore-Sigma catalog number 333539900) before filtration through a 40 μm pore size Flowmi or Plurisselect filter at the top of a 50mL falcon tube and the sample was aspirated through the filter using a large plunger. The filtered sample was further centrifuged at 500rcf for 5 minutes and resuspended in buffer to provide the final dissociated fixed cell sample.

Results: dissociated fixed cells in final samples prepared from minced-fixed brain tissue can be visualized and counted using SYTO RNA dye (1:1000) and/or ethidium homodimer (1:1, after each aliquot was resuspended in 200 μl of water stock solution) all maintained at-20 ℃. Trypan blue can be used to more easily visualize any remaining debris.

Example 2: analysis of single cell 3' sequences of minced-fixed mouse kidney tissue and brain tissue

This example illustrates the preparation of a mouse kidney tissue sample and brain tissue sample using a chopper-fixation method for comparative study of single cell 3' sequence analysis up to 7 days after fixation.

Materials and methods:

A. preparing a sample:

minced-fixed cell samples were prepared from mouse kidney tissue and mouse brain tissue as described in example 1.

In addition, the following control samples were prepared: (1) Suspended PBMCs (not from tissue) fixed with 4% PFA; (2) Instead of being minced, the tissue is simply treated with a dissociating agent and then fixed in 4% pfa, whereby kidney tissue prepared therefrom immobilizes cells; (3) Fresh control PBMCs thawed from cryopreservation using RPMI and 10% fbs; (4) Fresh control kidney tissue samples (not fixed) were prepared by shredding fresh kidney tissue followed by incubation with a mixture of 2.5mg/mL collagenase a (Roche) and 10mg/mL pancreatic juice (Sigma, P1625) for 30 minutes at 37 ℃; and (5) B27-free dissociation (Brainhibit) from adult mice by chopping fresh brain tissue at 30 ℃ ^TM 2mg/mL papain of (E)E-Ca (HE-Ca) or +.>A-Ca (HA-Ca) solution was incubated for 20 minutes, then washed twice with Hibernate and resuspended with Nbactiv1 (Brainhibit), and fresh control brain tissue samples (not fixed) were prepared.

B. Single cell 3' sequence analysis

Separation of the unfixed samples into GEM and 3' -RT

The pellet fraction collected from the unfixed/protease treatment was centrifuged at 300g for 5 min, washed twice with a PBS solution of 0.04% bsa, and then loaded into a single cell 3' v3 protocol standard master mix used with a chromoum System (10X Genomics,Pleasanton,CA,USA) for separating the sample along with the barcoded gel beads in discrete droplets called GEM ("gel beads in emulsion"). Once GEM is generated, it is collected and then subjected to a thermal incubation step. The heating step helps to release the cell contents and RNA, capture the RNA through the barcode oligonucleotide, and cause a Reverse Transcription (RT) reaction that synthesizes cDNA to incorporate the barcode into the 3' synthon. cDNA electropherograms were performed using an Agilent 2100 bioanalyzer 5067-4626 to assess DNA size and yield for each sample.

Results

As shown by the results depicted in the comparison graphs of fig. 14A and 14B, the cut-fixed kidney tissue sample preparation exhibited a higher percentage of detected genes at day 1 relative to day 6 with only minor degradation relative to the "post-dissociation fixed" kidney tissue sample. Furthermore, as shown by the results depicted in the graph of fig. 14C, the minced-fixed brain tissue showed a significant increase in both detected genes and UMI relative to the fresh brain tissue sample.

Example 3: ATAC sequencing and Gene expression analysis of minced-fixed mouse brain tissue

This example illustrates the preparation of mouse brain tissue samples using the chopper-immobilized method for analysis using a series of single cell/single cell nuclear analysis methods, including ATAC analysis.

Materials and methods:

A. preparing a sample:

minced-fixed cell samples were prepared from mouse brain tissue as described in example 1. A "fresh control" brain tissue sample was prepared as described in example 2. The brain tissue samples "fixed after dissociation" were prepared without shredding, but the tissues were simply treated with dissociating agents, then fixed in 4% PFA.

B. Single cell multiunit analysis：

Brain tissue nuclei were prepared in three different ways: 1) Isolating fresh nuclei directly from E18 brain tissue; 2) Fixing fresh nuclei from E18 brain tissue and then treating with a unfixed agent of compound (8) as described above for the preparation of fixed cell samples; or 3) extracting the cut-fixed E18 brain tissue using the same method as the fresh nuclear sample to obtain fixed nuclei, followed by treatment with the unfixed agent of compound (8). The three brain tissue preparations were loaded into a chromasum instrument (10 x Genomics), treated with single Cell multicellular multigang atac+ gene expression solutions (10 x Genomics), and analyzed using a Cell range ARC pipeline (10 x Genomics), including single Cell/single Cell nuclear 3' gene expression paired with ATAC sequencing.

Results

As shown by the results summarized in table 2 (below), the "cut-to-fix post-separate" E18 brain tissue sample preparation showed significantly higher percentages of genes and UMI detected (relative to fresh control samples) relative to the "post-dissociation fixed" brain tissue sample. In addition, the minced-fixed brain tissue preparations exhibited significantly higher percentages of unique ATAC fragments than the "post-dissociation fixed" or even fresh control samples.

TABLE 2

Example 4: single cell multi-set analysis of post-fixation de-fixed mouse brain tissue

This example illustrates a study in which a sample of mouse brain tissue was prepared using a chopper-immobilized method, then the sample was deblocked using a deblocker compound (8), followed by analysis of the deblocked sample using a series of single cell analysis methods, including ATAC analysis.

Materials and methods:

A. preparation of fixed cell samples:

minced-fixed cell samples were prepared from mouse brain tissue as described in example 1. A "fresh nucleus" brain tissue sample was prepared in the same manner as the "fresh control" sample described in example 2.

B. Preparation of the Defixative Compound (8)

The following two-step synthetic procedure was used to prepare the unfixed agent compound (8).

Step 1: (4-aminopyridin-3-yl) phosphonic acid diethyl ester. In step 1, the compound diethyl (4-aminopyridin-3-yl) phosphonate was prepared according to the procedure described in Guilar, R.et al, synthesis,2008,10,1575-1579. Briefly, diethyl phosphite (2.2 mL,17.3mmol,1.2 eq), triethylamine (3 mL,1.5 eq), PPh3 (1.1 g,4.3mmol,30 mol%) and Pd (OAc) 2 (0.39 g,1.73mmol,12 mol%) were added to a solution of 3-bromopyridin-4-amine (2.5 g,14.5mmol,1 eq.) in ethanol (58 mL) (CAS number 13534-98-0, sigma Aldrich). The reaction mixture was purged with argon for 5min. After heating to reflux for 24 hours, the reaction mixture was cooled to room temperature and concentrated in vacuo. The residue was purified by silica gel chromatography (MeOH/DCM) to give the title compound (0.35 g, 11% yield). 1H NMR (80 MHz, CDCl 3): delta=1.15 (t, 6H, CH 3), 4.18-3.69 (m, 4H, CH 2), 5.99 (br-s, 2H, NH 2), 6.49 (d, 1H), 8.03-7.93 (m, 1H), 8.22 (d, 1H).

Step 2: (4-aminopyridin-3-yl) phosphonic acid (compound (8)). In step 2, the target compound (4-aminopyridin-3-yl) phosphonic acid (compound (8)) is prepared by acid hydrolysis of the step 1 precursor compound. Diethyl (4-aminopyridin-3-yl) phosphonate (0.35 g,1.52mmol,1 eq.) was suspended in 6N HCl (aq) (8 mL). After refluxing for 12 hours, the reaction mixture was concentrated in vacuo. The residue was washed with DCM, diethyl ether and concentrated in vacuo to give the title compound (8) (247 mg, yield 93%). 1H NMR (80 MHz, D2O) delta=6.85-6.55 (m, 1H), 8.05-7.94 (m, 1H), 8.40-8.26 (m, 1H).

A stock solution of 300mM of compound (8) deammobilizer in 50mM Tris-HCl, 1mM EDTA (pH 8.3) was prepared, filtered using a 5 μm syringe filter and stored at room temperature.

C. Sample de-fixation treatment

To the minced-fixed cell solution of step A, a ribonuclease inhibitor was added, along with 100mM of the deammobilizer compound (8), along with 10U of cold active protease, arcticzymes protease. The unfixed mixture was incubated at 8℃for 2 hours, followed by 15 minutes at a higher temperature of 70 ℃. The resulting unfixed cell solution was centrifuged at 500g at 4℃for 5 minutes, and the supernatant and the pellet fraction were collected, respectively. The "fresh nucleus" sample was not treated with any unfixed agent or protease.

D. Single cell multiunit analysis：

Brain tissue nuclei were prepared in three different ways: 1) Isolating fresh nuclei directly from E18 brain tissue; 2) Fixing fresh nuclei from E18 brain tissue and then treating with a unfixed agent of compound (8) as described above for the preparation of fixed cell samples; or 3) extracting the cut-fixed E18 brain tissue using the same method as the fresh nuclear sample to obtain fixed nuclei, followed by treatment with the unfixed agent of compound (8). The three brain tissue preparations were loaded into a chromasum instrument (10 x Genomics), treated with single Cell multicellular multigang atac+ gene expression solutions (10 x Genomics), and analyzed using a Cell range ARC pipeline (10 x Genomics), including single Cell/single Cell nuclear 3' gene expression paired with ATAC sequencing. Single gene expression and ATAC metrics were calculated, as well as combined RNA/ATAC sequencing metrics, as shown in fig. 15A and 15B.

Results

As shown by the results depicted in the comparison graphs of fig. 15A and 15B, ATAC transposition events from the minced-fixed brain tissue nucleus sample preparation unfixed with the unfixed agent compound (8) exhibited cleaner separation relative to transposition events from the "fresh nucleus" sample. This separation observed for the unfixed cut-fixed brain tissue corresponds to only 31% of the ATAC fragments overlapping the so-called peaks, while 55% of the "fresh nucleus" fragments overlapping the other so-called peaks. These results indicate that the minced-immobilized samples, which were unfixed using the unfixed agent, can provide higher quality ATAC data from their nuclei than can be obtained from fresh cells.

Example 5: analysis of Single cell 3' sequences of the mouse kidney tissue unfixed after minced-fixed

This example illustrates the preparation of a mouse kidney tissue sample using a chopper-immobilized method, followed by the use of a unfixed agent compound (8) to unfixed the sample, followed by the study of single cell 3' sequence analysis of the unfixed sample.

Materials and methods:

A. preparation of fixed cell samples:

minced-fixed cell samples were prepared from mouse kidney tissue as described in example 1. A "fresh control" kidney tissue sample was prepared as described in example 2.

B. Sample de-fixation treatment

The minced-fixed kidney tissue was stored in PBS + ribonuclease inhibitor and 0.04% BSA for up to 6 days. On the day of analysis, the fixed kidney tissue samples were dissociated, and then the samples in step a were subjected to a deblocking treatment using the deblocking treatment described in example 4, followed by analysis by single cell 3' gene expression.

C. Single cell 3' sequence analysis

Single cell 3' sequence analysis of the unfixed samples was performed as described in example 2.

D. Cell count

Cell types present in the cut-fixed kidney tissue samples that were unfixed with compound (8) were determined and mapped by automated meta-analysis using cell clusters identified by differential expressed marker gene expression. The composition of tissue cell types is identified by an automated script that quantifies the number and fraction of cell types known to be detected in kidney tissue samples by classifying the cells based on a combination of differentially expressed known marker genes for each cell type, wherein unclassified cells fall into an unclassified class.

Results

As shown by the results summarized in table 3 (below), it was found that the minced-fixed, dissociated, and stored kidney tissue for up to 6 days showed reasonable sensitivity in various single cell 3' sequence analysis metrics relative to cells from freshly dissociated kidney tissue. Single cell sequencing data was downsampled to normalize for sequencing depth differences at 30,000 raw reads per cell (rrpc) depth.

TABLE 3 Table 3

As shown by the results depicted in the graph of fig. 16, it was found that kidney tissue minced-fixed, dissociated, de-fixed, and stored for up to 6 days exhibited more robust clusters of cell types compared to fresh tissue samples, including the presence of 15 larger clusters out of 24 clusters, including the presence of more of the following cell types: distal tubular cells, collecting duct cells and interstitial cells. In addition, the minced-post-fixed unfixed kidney tissue samples exhibited minimal heat shock and cellular stress gene signatures (Jun, hspa4, gadd45b and Nr4a 1) compared to fresh cells treated with collagenase digestion at 37 ℃.

Example 6: analysis of Single cell 3' sequences of the mouse brain tissue after the chop-fixation

This example illustrates the study of preparing a mouse brain tissue sample using a chopper-immobilized method, then using a unfixed agent compound (8) to unfixed the sample, followed by single cell 3' sequence analysis of the unfixed sample.

Materials and methods:

A. preparation of fixed cell samples:

minced-fixed cell samples were prepared from mouse brain tissue as described in example 1.

B. Sample de-fixation treatment

The unfixed treatment for the fixed brain tissue sample in step a was performed using the unfixed treatment described in example 4.

C. Single cell 3' sequence analysis

D. Cell count

The cell types present in the minced-fixed brain tissue samples unfixed with compound (8) were determined and mapped in the same manner as described for kidneys in example 5.

Results

As shown by the results summarized in table 4 (below), it was found that the minced-fixed, dissociated, and stored brain tissue for up to 6 days showed greatly improved sensitivity in various single cell 3' sequence analysis metrics relative to cells from freshly dissociated brain tissue.

TABLE 4 Table 4

Cell type count and profiling were further performed on the brain cell types present in the samples and the results are summarized in table 5 (below).

TABLE 5

The results summarized in table 5 show that genes from neurons and UMI are more prevalent in the post-chopper-immobilized unfixed samples than in the fresh samples, whereas endothelial cells and glial cells are present in brain tissue more prevalent in the fresh samples. The minced-fixed brain tissue preparations are not only more neuronal but also exhibit up to 3-fold complexity. Furthermore, similar to the samples from kidney tissue, the cell samples prepared from the minced-fixed brain tissue showed far fewer heat-induced stress genes than freshly dissociated cells (Jun, fox, dusp and Nr4a 1).

Example 7: cut-to-fix method for preparing human uterine tissue for single cell sequence analysis

This example illustrates the preparation of human uterine tissue samples using the cut-and-fix method for further analysis using single cell RNA-based sequencing methods.

Materials and methods

Fresh tissue is collected from normal uterine tissue adjacent to tissue exhibiting endometrial cancer. Tissue was surgically removed and covered in ice for laboratory analysis within 16 to 24 hours after surgery. However, transport delays result in tissue being received by the laboratory about 48 hours after surgery, when the tissue is at room temperature.

The tissue is cut into smaller tissue pieces, which are then subjected to seven different cut-fixation methods and/or other preparation methods for downstream single cell assays as described below.

The cut-fixation of tissue sections was performed as described above in examples 1 to 6. After chopping, the tissue was fixed in 4% paraformaldehyde solution at 4 ℃ for 16 to 24 hours. After fixation, the minced-fixed tissue was centrifuged at 850rcf for 5 min at room temperature and the supernatant removed without disturbing the fixed tissue pellet. The fixed tissue pellet was dissociated as described below.

80. Mu.L of Liberase stock solution was added to 1,920. Mu.L of RPMI and mixed to prepare a dissociation solution of RPMI+0.2mg/mL Liberase. The dissociation solution was stored at 4℃and pre-heated at 37℃for 10 minutes before use.

2mL of the pre-heated dissociation solution was added to the fixed tissue sample. The sample was dissociated using an Octo dissociator or manual dissociation as described below.

Octo dissociator: samples were transferred to Miltenyi C tubes. The use of these C-tubes requires a minimum of 2mL of dissociation solution. The C tube was placed in an Octo-dissociator and the following procedure was run: incubation at 37℃for 20 min at 50 rpm; clockwise rotation at 2,000rpm for 30 seconds at 37 ℃; at 37℃the rotation was counter-clockwise at 2,000rpm for 30 seconds. The C-tube was separated and the dissociated tissue was passed through a 70 μm filter to remove debris and undissociated tissue pieces.

Manual dissociation: the samples were incubated at 37℃for 20 minutes and the tubes were shaken intermittently. Tissue pieces were ground 15 to 20 times (until the solution began to become cloudy) using a silanized glass pipette to obtain a single cell suspension. The dissociated tissue is passed through a 70 μm filter to remove debris and undissociated tissue pieces.

After either dissociation protocol, the samples were centrifuged at 850rcf for 5 minutes and the supernatant removed without disturbing the pellet. The pellet was resuspended in 1mL or chilled buffer.

For this comparative study, a total of seven different uterine tissue samples were prepared and analyzed: (1) Tissues dissociated into single cells and without any fixation, then subjected to a 10x Genomics single cell 3' v3 assay (data not shown); (2) Tissues dissociated into single cells and then subjected to 10x Genomics fixed RNA spectroscopy assay (results shown in fig. 29A); (3) Quick-freezing followed by treatment with a 10x Genomics nuclear separation kit followed by subjecting the separated nuclei to 10x Genomics fixed RNA spectroscopy assay of fresh tissue (results shown in fig. 29B); (4) Cut-fixed, then dissociated into single-cell fresh tissue (results shown in fig. 29C); (5) After quick freezing, the pieces were minced-fixed and then dissociated into single-cell fresh tissue (the results are shown in fig. 29D); (6) Cut-fixed, then stored at 4 ℃ for 5 days, then dissociated into single cells and subjected to 10x Genomics fixed RNA spectroscopy assay of fresh tissue (results shown in fig. 29E); and (7) fresh tissue that was cut-fixed after quick-freezing, then stored at 4 ℃ for 5 days, then dissociated into single cells and subjected to 10x Genomics fixed RNA spectroscopy assay (results shown in fig. 29F).

Results

Fig. 29A-29F depict graphs of cell type clusters detected in these seven preparations of human uterine tissue samples. It should be noted that cells prepared without any fixation and assayed using the standard 10x Genomics single cell 3' v3 assay did not generate any data that could be used to prepare such a cell cluster map. Comparison of these figures shows that at least nine different cell types can be identified using the RNA profiling assay utilized in the present study, however, the detection results for certain cell type clusters vary widely. As is extremely clear from a comparison of the graphs of fig. 29A-29F, many cell types exhibited either an deficiency or absence in fresh dissociated samples subjected to the standard of 10x Genomics RNA spectroscopy assay (fig. 29A) when compared to the samples dissociated after shredding-fixation and subjected to the whole cell 10x Genomics fixed RNA spectroscopy assay (fig. 29C-29F). The most obvious deletion is in stromal cells, which fall within outlined region "1" in each of the figures 29A-29F. In addition, as shown by the results depicted in fig. 29E and 29F, human uterine tissue can be minced-fixed and stored at 4 ℃ for 5 days, after which the minced-fixed tissue is enzymatically dissociated and subjected to single cell RTL analysis. The data obtained from cells stored for 5 days showed no loss in sample complexity compared to the sample for the immediate day of treatment.

Overall, the single cell samples provided by the cut-to-fix method were able to return enough data to interpret/identify multiple cell types in the samples, whereas single cell samples prepared without fixation resulted in complete failure to generate RNA data, and were prepared using tissue dissociation alone (as is common in tissue processing laboratories), after which single cell samples subjected to 10x Genomics fixed RNA spectroscopy analysis showed very poor results with respect to the number of RNA transcripts and ability to differentiate between different cell types (see fig. 29A versus fig. 29C-29F). These results demonstrate that the shredder-immobilization methods described herein can be used to prepare human tissue samples suitable for single cell RNA spectroscopy assays, even from tissue samples that are under less than ideal conditions due to transport delays. Thus, the chopper-immobilized method for preparing tissue samples enables the preparation of samples that can more easily withstand generally adverse transport (or storage) times and conditions, allowing for advantageous single cell analysis of transported and/or stored samples at a remote location.

Although the foregoing disclosure has been described in some detail by way of illustration and description for purposes of clarity and understanding, the disclosure including examples, detailed description and embodiments described herein is intended to be illustrative and should not be construed as limiting the disclosure. It will be apparent to those skilled in the art that various modifications or changes to the examples, descriptions and embodiments described herein may be made and are intended to be included within the spirit and scope of the disclosure and the appended claims. Furthermore, those skilled in the art will recognize many methods and programs that are equivalent to those described herein. All such equivalents are understood to be within the scope of this disclosure and are covered by the appended claims.

Additional embodiments of the present disclosure are set forth in the following claims.

The disclosures of all publications, patent applications, patents, or other documents mentioned herein are expressly incorporated by reference in their entirety for all purposes to the same extent as if each such individual publication, patent application, or other document was specifically and individually indicated to be incorporated by reference in its entirety for all purposes and were set forth fully herein. In case of conflict, the present specification, including the terms specified, will control.

Claims

1. A method of preparing a biological sample, comprising:

(a) Chopping biological tissue into a tissue fragment composition;

(c) Treating the fixed tissue fragment composition with a cell dissociation agent, thereby providing a fixed cell composition.

2. The method of claim 1, wherein the tissue fragment composition comprises particles having an average size on one side of about 500 μιη or less, about 250 μιη or less, about 125 μιη or less, about 75 μιη or less, or about 50 μιη or less.

3. The method of any one of claims 1-2, wherein the tissue fragment composition comprises particles having an average size on one side of between about 50 μιη and 500 μιη, between about 125 μιη and 500 μιη, between about 250 μιη and 500 μιη, between about 50 μιη and 250 μιη, or between about 50 μιη and 125 μιη.

4. A method according to any one of claims 1 to 3, wherein the fixing agent is paraformaldehyde ("PFA"); optionally, wherein the PFA is present in the solution at a concentration of 1% to 4%.

5. The method of any one of claims 1 to 4, wherein the cell dissociation agent comprises collagenase.

6. The method of any one of claims 1-5, wherein the amount of time before treating the fixed tissue fragment with the cell dissociation agent is at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 1 week, at least 1 month, at least 6 months, or more.

7. The method of any one of claims 1 to 6, wherein the fixed cell composition comprises a plurality of fixed cell types.

8. The method of any one of claims 1 to 7, wherein the immobilized cells of the immobilized cell composition comprise a plurality of crosslinked nucleic acid molecules.

9. The method of any one of claims 1 to 8, wherein the method further comprises:

(a) Treating the fixed cell composition with a solution comprising a unfixed agent, thereby providing a plurality of types of unfixed cells from the biological tissue.

10. The method of claim 9, wherein the unfixed agent is capable of removing crosslinks formed in the biomolecule by fixation with 1% to 4% strength paraformaldehyde ("PFA") solution.

11. The method of any one of claims 9 to 10, wherein the unfixed agent comprises a compound selected from the group consisting of: compound (1), compound (2), compound (3), compound (4), compound (5), compound (6), compound (7), compound (8), compound (9), compound (10), compound (11), compound (12), compound (13), compound (14), compound (15), or a combination thereof.

12. The method of claim 11, wherein the concentration of the deammobilizing agent is about 1mM to about 500mM, about 50mM to about 300mM, or about 50mM to about 200mM.

13. The method of any one of claims 9 to 12, wherein the solution comprising a unfixed agent further comprises a protease.

14. The method of claim 13, wherein the protease is a thermolabile protease or a cold-active protease; optionally, wherein the protease is selected from the group consisting of subtilisin a, protease K, arcticZymes protease, and combinations thereof.

15. The method of any one of claims 1 to 14, wherein the biological tissue is selected from brain tissue, skin tissue, muscle tissue, smooth muscle tissue, cardiac muscle tissue, skeletal muscle tissue, bone marrow tissue, lung tissue, bronchus tissue, oviduct tissue, gall bladder tissue, ovary tissue, testis tissue, hypothalamus tissue, thyroid tissue, adrenal tissue, kidney tissue, pancreas tissue, small intestine tissue, large intestine tissue, colon tissue, liver tissue, lymph tissue, breast tissue, mesenteric tissue, nasal tissue, pineal tissue, parathyroid tissue, pharyngeal tissue, laryngeal tissue, pituitary tissue, prostate tissue, saliva tissue, spinal cord tissue, spleen tissue, stomach tissue, thymus tissue, tracheal tissue, tongue tissue, urethra tissue, placenta tissue, artery tissue, vein tissue, and tonsil tissue.

16. The method of any one of claims 1 to 15, wherein the method further comprises filtering and/or sieving the fixed cell composition and/or the unfixed cells.

17. A method of analyzing biological tissue, comprising

(a) Chopping biological tissue to provide a tissue fragment composition;

(c) Treating the fixed tissue fragment composition with a cell dissociation agent to provide a fixed cell composition, each fixed cell comprising a plurality of crosslinked nucleic acid molecules; and

18. The method of claim 17, wherein the generating a plurality of barcoded nucleic acid molecules is performed in a plurality of partitions; optionally, wherein the plurality of partitions is a plurality of droplets or holes.

19. The method of claim 18, wherein one of the plurality of partitions comprises immobilized cells and a support comprising the plurality of nucleic acid barcode molecules.

20. The method of claim 19, wherein the support is a bead.

21. The method of any one of claims 18 to 20, wherein the barcode sequence is a partition specific barcode sequence.

22. The method of any one of claims 17 to 21, wherein the tissue fragment composition comprises particles having an average size on one side of about 500 μιη or less, about 250 μιη or less, about 125 μιη or less, about 75 μιη or less, or about 50 μιη or less.

23. The method of any one of claims 17-22, wherein the tissue fragment composition comprises particles having an average size on one side of between about 50 μιη and 500 μιη, between about 125 μιη and 500 μιη, between about 250 μιη and 500 μιη, between about 50 μιη and 250 μιη, or between about 50 μιη and 125 μιη.

24. The method of any one of claims 17 to 23, wherein the immobilization reagent is paraformaldehyde ("PFA"); optionally, wherein the PFA is present in the solution at a concentration of 1% to 4%.

25. The method of any one of claims 17 to 24, wherein the cell dissociation agent comprises collagenase.

26. The method of any one of claims 17 to 25, wherein the amount of time before treating the fixed tissue fragment with the cell dissociation agent is at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 1 week, at least 1 month, at least 6 months, or more.

27. The method of any one of claims 17 to 26, wherein the fixed cell composition comprises a plurality of fixed cell types.

28. The method of any one of claims 17 to 27, wherein the fixed cells of the fixed cell composition comprise a plurality of crosslinked nucleic acid molecules.

29. The method of any one of claims 17 to 28, wherein the biological tissue is selected from brain tissue, skin tissue, muscle tissue, smooth muscle tissue, cardiac muscle tissue, skeletal muscle tissue, bone marrow tissue, lung tissue, bronchus tissue, oviduct tissue, gall bladder tissue, ovary tissue, testis tissue, hypothalamus tissue, thyroid tissue, adrenal tissue, kidney tissue, pancreas tissue, small intestine tissue, large intestine tissue, colon tissue, liver tissue, lymph tissue, breast tissue, mesenteric tissue, nasal tissue, pineal tissue, parathyroid tissue, pharyngeal tissue, laryngeal tissue, pituitary tissue, prostate tissue, saliva tissue, spinal cord tissue, spleen tissue, stomach tissue, thymus tissue, tracheal tissue, tongue tissue, urethra tissue, placenta tissue, artery tissue, vein tissue, and tonsil tissue.

30. The method of any one of claims 17 to 29, wherein the method further comprises filtering and/or sieving the fixed cell composition and/or the unfixed cells.

31. A method of analyzing biological tissue, comprising

(a) Chopping biological tissue to provide a tissue fragment composition;

(c) Treating the fixed tissue fragment composition with a cell dissociation agent to provide a fixed cell composition, each fixed cell comprising a plurality of crosslinked nucleic acid molecules;

(d) Treating the fixed cell composition with a defreezing agent to provide a defreezed cell composition, each defreezed cell comprising a plurality of uncrosslinked nucleic acid molecules; and

32. The method of claim 31, wherein the generating a plurality of barcoded nucleic acid molecules is performed in a plurality of partitions; optionally, wherein the plurality of partitions is a plurality of droplets or holes.

33. The method of any one of claims 31 to 32, wherein treating with the unfixed agent is performed in a plurality of partitions.

34. The method of any one of claims 31 to 33, wherein the plurality of partitions are a plurality of droplets or wells.

35. The method of any one of claims 31-34, wherein one partition of the plurality of partitions comprises a disarmed cell and a support comprising the plurality of nucleic acid barcode molecules.

36. The method of claim 34, wherein the support is a bead.

37. The method of any one of claims 31-36, wherein the barcode sequence is a partition-specific barcode sequence.

38. The method of any one of claims 31-37, wherein the tissue fragment composition comprises particles having an average size on one side of about 500 μιη or less, about 250 μιη or less, about 125 μιη or less, about 75 μιη or less, or about 50 μιη or less.

39. The method of any one of claims 31-38, wherein the tissue fragment composition comprises particles having an average size on one side of between about 50 μιη and 500 μιη, between about 125 μιη and 500 μιη, between about 250 μιη and 500 μιη, between about 50 μιη and 250 μιη, or between about 50 μιη and 125 μιη.

40. The method of any one of claims 31 to 39, wherein the immobilization reagent is paraformaldehyde ("PFA"); optionally, wherein the PFA is present in the solution at a concentration of 1% to 4%.

41. The method of any one of claims 31 to 40, wherein the cell dissociation agent comprises collagenase.

42. The method of any one of claims 31-41, wherein the amount of time before treating the fixed tissue fragment with the cell dissociation agent is at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 1 week, at least 1 month, at least 6 months, or more.

43. The method of any one of claims 31-42, wherein the fixed cell composition comprises a plurality of fixed cell types.

44. The method of any one of claims 31 to 43, wherein the immobilized cells of the immobilized cell composition comprise a plurality of crosslinked nucleic acid molecules.

45. The method of any one of claims 31 to 44, wherein the biological tissue is selected from the group consisting of bone, brain, colon, epithelium, kidney, liver, lung, muscle, ovary, pancreas, and uterus.

46. The method of any one of claims 31 to 45, wherein the method further comprises filtering and/or sieving the fixed cell composition and/or the unfixed cells.

47. A kit, comprising: immobilization reagents, cell dissociation reagents and assay reagents.

48. The kit of claim 47, wherein the kit further comprises a unfixed agent.

49. The kit of any one of claims 47 to 48, wherein the kit further comprises a shredding device.

50. A morcellating device for mechanically morcellating a biological tissue sample, the device comprising:

(a) A razor cartridge comprising a plurality of razor blade slots, each slot configured to insert a blade edge of a razor blade through the razor blade slot and retain a top portion of the razor blade within the razor blade slot, wherein a bottom portion of the razor blade comprising the razor blade edge extends vertically below the razor cartridge;

(b) A razor blade alignment layer comprising a plurality of alignment openings, wherein each alignment opening is configured to insert the blade edge of the razor blade through the alignment layer such that the bottom portion of the razor blade comprising the razor blade edge extends below the alignment layer; and

wherein the razor cartridge is configured to be placed over and connected to the razor blade alignment layer such that each of the plurality of razor blade slots is aligned over each of a corresponding plurality of alignment openings; and is also provided with

51. The device of claim 50, wherein the razor cartridge comprises at least two through holes and at least two screws inserted through the at least two through holes for reversibly connecting the razor cartridge to the razor blade alignment layer.

52. The device of claim 51, wherein each of the at least two screws is a spring-loaded screw configured to provide a vertical lifting force to the razor cartridge relative to the alignment layer so as to provide a collapsible gap between the razor cartridge and the alignment layer.

53. The device of claim 52, wherein the spring-loaded screw is configured such that the gap between the bottom of the razor cartridge and the top of the alignment layer is in the range of 0.5mm to 10mm when at rest.

54. The device of claim 50, further comprising a handle connected to a top of the razor cartridge.

55. The device of claim 54, wherein the handle includes at least one through hole for a screw to pass through to connect the handle to the razor cartridge.

56. The device of claim 50 wherein said razor cartridge has a cartridge diameter, said plurality of razor cartridge slots being parallel and having a length of at least 85% of said cartridge diameter.

57. The device of claim 50 wherein said razor blade alignment layer has an alignment layer diameter, said plurality of alignment openings being parallel and having a length of at least 75% of said frame diameter.

58. The apparatus of claim 50, wherein the circular sample disk holder is a bowl configured to hold a sample disk.

59. The device of claim 58 wherein the razor cartridge and the razor blade alignment layer are configured such that the blade edge of each razor blade inserted into the slot and the opening will contact the bottom of the sample tray when the razor cartridge and the razor blade alignment layer are pushed downward.

60. The apparatus of claim 50, wherein the circular sample disk holder comprises a top circular edge, the alignment layer comprising an annular channel around an outer perimeter of a circular bottom surface and corresponding to the top circular edge of the sample disk holder, wherein the annular channel is configured to receive the top circular edge to reversibly and rotatably couple the pair Ji Ceng to the circular sample disk holder.

61. The apparatus of claim 60, wherein the alignment layer comprises a second annular channel around an outer perimeter of the circular bottom surface and within an inner perimeter of the annular channel, wherein the second annular channel is configured to receive a top circular edge of a sample disk held within the circular sample disk holder.

62. The device of claim 50, wherein each razor blade slot opening ranges from 35mm to 45mm in length and from 0.1mm to 0.6mm in width.

63. The apparatus of claim 50, further comprising a sample tray.

64. A method of using the morcellating device of claim 50, wherein the device includes a plurality of razor blades inserted into the plurality of razor blade slots and the plurality of aligned openings, placed over and reversibly and rotatably connected to the circular sample disk holder, the holder containing an open circular sample disk containing a tissue sample, the method comprising:

(i) Pressing down on the razor cartridge, thereby forcing the blade edges of the plurality of razor blades against and cutting the tissue sample contained within the sample tray;

(iii) Pressing down on the razor cartridge, thereby forcing the cutting edges of the plurality of razor blades to press against and cut the tissue sample in the second orientation.

65. The method of claim 64, further comprising rotating the plurality of razor blades to a third orientation relative to the tissue sample and then pressing the razor cartridge downward onto the circular sample tray holder, thereby forcing the cutting edges of the plurality of razor blades to press onto and cut the tissue sample in the third orientation.

66. The method of claim 65, further comprising rotating the plurality of razor blades to a fourth orientation relative to the tissue sample and then pressing the razor cartridge downward onto the circular sample tray holder, thereby forcing the cutting edges of the plurality of razor blades to press onto and cut the tissue sample in the fourth orientation.

67. The method of claim 64 wherein the plurality of razor blades rotate at least 10 to 25 degrees per rotation.

68. A kit comprising the morcellating device of claim 50, and one or more razor blades in one or more containers.