CN113026110A - High-throughput single-cell transcriptome sequencing method and kit - Google Patents
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- C—CHEMISTRY; METALLURGY
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- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The invention discloses a high-throughput single-cell transcriptome sequencing method, which comprises the steps of single-cell capture and packaging by utilizing a microfluidic chip, RAN reverse transcription, cDNA pre-amplification, primary amplification, secondary amplification, fragmentation, library construction, sequencing, analysis and the like. The sequencing method has the advantages of high cell capture efficiency, high flux (up to 1 ten thousand cells), high flexibility (1-8 samples can be made at the same time), low cross contamination, low double-package rate, simple operation, low cost and the like, and has wide application prospects in a plurality of fields of environment, infectious diseases, rejection after organ transplantation, immunotherapy and the like.
Description
Technical Field
The invention relates to a gene sequencing method, in particular to a novel high-throughput single-cell transcriptome sequencing method and a corresponding kit, belonging to the field of molecular biology.
Background
The single cell transcriptome can detect the heterogeneity of single cells and provide accurate information for the diagnosis and treatment of diseases. Due to the problems of processing flux, cost and the like of the single cell sequencing technology, a lot of large-scale single cell epigenetic research work cannot be carried out. For example, one conventional approach is to use a pipette for micromanipulation, which is low throughput, time consuming, and tedious; another way is to use flow cytometry, which requires a large sample volume, requires precise control, and is damaging to the cells. The requirement for the subsequent library building is high.
It is speculated that the combination of microfluidic technology and single cell methylation technology may solve these problems well. Based on such expectations, researchers have proposed various approaches. For example, the U.S. Fludigm company introduced in 2014 a C1 single cell fully automated system that utilizes microvalves to achieve single cell separation with throughput that has been increased to the point where hundreds of single cells can be analyzed at a time. The advent of this system has made it possible to study single cell methylation on a large scale. Meanwhile, due to the visualization operation, more than two cells of the sample can be removed. As another example, the manufacturer proposed a Microwell-split pool system, which allows the isolation of single cells. For another example, manufacturers developed 10X genomics and Biorad systems that use microfluidic chips to encapsulate labeled microbeads and single cells in a droplet to achieve single cell separation and labeling. The flux can reach 1 ten thousand cells. However, these techniques still suffer from more or less some drawbacks, such as:
the aforementioned C1 system has low throughput, can only make 938 cells at most, and is costly. The throughput of the above Microwell-split pool system is also low, usually 1000-2000 cells, the reaction volume is large, the reagent consumption is high, the cost is high and the cell loss is large. In the 10X genomics and Biorad systems, the droplet generating device needs to be driven by electric power, which is costly, and only 4 or 8 samples can be simultaneously made in each experiment, so that the flexibility is limited.
Disclosure of Invention
The invention mainly aims to provide a high-throughput single-cell transcriptome sequencing library, a high-throughput single-cell transcriptome sequencing method and a corresponding kit, thereby overcoming the defects of the prior art.
In order to achieve the aim of the invention, the invention adopts the following scheme:
the embodiment of the invention provides a method for constructing a high-throughput single-cell transcriptome sequencing library, which comprises the following steps:
capturing and packaging single cells by using a microfluidic chip so as to generate water-in-oil reaction droplets containing single cells;
cracking and reverse transcribing the cells in the water-in-oil reaction liquid drop to obtain a reverse transcription product;
performing demulsification treatment on the water-in-oil reaction liquid drop, and performing pre-amplification treatment on the reverse transcription product to obtain a pre-amplification product;
and (3) carrying out fragmentation treatment on the pre-amplification product, and then adding adapters at two ends of the obtained fragmentation product to generate a sequencing library.
In some embodiments, the construction method specifically includes:
providing a micro-fluidic chip which comprises a cell micro-channel, a cell isolation medium micro-channel, a cell label micro-channel and a single-cell sample collecting port;
respectively injecting the cell suspension and the cell label suspension into the microfluidic chip, and mixing the cell suspension and the cell label suspension to form cell carrier liquid after the cell suspension and the cell label suspension respectively flow through the cell microchannel and the cell label microchannel;
injecting a cell isolation medium into the microfluidic chip, enabling the cell isolation medium to be in contact with the cell carrier liquid when flowing in the cell isolation medium microchannel, and shearing and wrapping the cell carrier liquid to form a water-in-oil reaction droplet containing a single cell and a single cell label;
and collecting the water-in-oil reaction droplets from the single cell sample collection port.
In some embodiments, the water-in-oil reaction droplet further comprises a cell lysis reagent and an RNA reverse transcription reagent.
In some embodiments, the cell tag suspension comprises a deformable microbead and a polyT primer carrying a tag, the polyT primer is attached to the deformable microbead, and the polyT primer is capable of detaching from the deformable microbead under physical and/or chemical action.
The embodiment of the invention also provides a high-throughput single-cell transcriptome sequencing method, which comprises the following steps: a single cell transcriptome sequencing library was constructed using any of the methods described previously, followed by sequencing analysis.
The embodiment of the invention also provides a kit for constructing the high-throughput single-cell transcriptome sequencing library, which comprises the following components:
a microfluidic chip for capturing at least single cells and packaging to generate a water-in-oil reaction droplet, the water-in-oil reaction droplet comprising an oil phase and a cellular liquid phase encapsulated by the oil phase, and the water-in-oil reaction droplet comprising single cells, a single cell tag, a cell lysis reagent, and an RNA reverse transcription reagent;
an oil for forming the oil phase;
a cell lysis reagent;
an RNA reverse transcription reagent;
a cell tag comprising a deformable microbead and a tagged polyT primer attached to the deformable microbead, the tagged polyT primer capable of detaching from the deformable microbead under physical and/or chemical action; and
cDNA pre-amplification reagents, transposition fragmentation reagents and reagents required to attach linkers at both ends of the fragmentation product. .
In some embodiments, the microfluidic chip comprises a cell micro-channel, a cell isolation medium micro-channel, a cell label micro-channel and a single-cell sample collection port, the cell micro-flow channel is provided with a cell suspension inlet and a single cell suspension outlet, the cell isolation medium micro-flow channel is provided with a cell label suspension inlet and a cell label suspension outlet, and the single-cell suspension outlet is intersected with the cell label suspension outlet, so that the single-cell suspension output by the cell micro-channel can be mixed with the cell label suspension output by the cell label micro-channel to form cell carrier liquid, the flow path of the cell carrier liquid is intersected with the cell isolation medium micro-channel, so that the cell isolation medium flowing in the cell isolation medium micro-channel can be cut and wrapped with the cell carrier liquid, thereby forming a water-in-oil reaction droplet comprising a single cell which is output by the single cell sample collection port.
Furthermore, the tail region of the cell micro-channel is set as a single-cell channel, the width of the single-cell channel is equal to or slightly larger than the diameter of a single cell, the outlet of the single-cell channel intersects with the outlet of the cell label micro-channel, so that the single-cell suspension output from the cell micro-channel and the cell label suspension output from the cell label micro-channel are mixed to form a cell carrier liquid, the continuous flow path of the cell carrier liquid intersects with the cell isolation medium micro-channel, so that the cell isolation medium flowing through the cell isolation medium micro-channel can shear the continuous cell carrier liquid into discrete droplet-shaped cell liquid phase and enable each cell liquid phase to contain a single cell and a single cell label, and meanwhile, the cell isolation medium is used as an oil phase to wrap the cell liquid phase, so that the water-in-oil reaction liquid droplet is formed.
According to the invention, gel beads are adopted to construct cell labels, reverse transcription is carried out in water-in-oil reaction liquid drops, an air pump and the like are used as a power source to drive the generation of the water-in-oil reaction liquid drops, a plurality of samples can be simultaneously made, the capture efficiency of cells is effectively improved, the mutual pollution of the beads after the liquid drops are demulsified is reduced, the proportion of effective data is improved, the cost is reduced, the use mode is more flexible, the overall operation time is shortened to 7 hours from 30 hours compared with the existing indep and hydropseq platforms, and the operation is easier.
In summary, compared with the prior art, the high-throughput single-cell transcriptome sequencing method provided by the invention improves the cell flux and reduces the cost, the flux is equivalent to the highest 10 Xgenomics in the current market, the single experiment can reach 2000-.
Drawings
FIG. 1 is a process flow diagram of a method for sequencing a high throughput single-cell transcriptome in an exemplary embodiment of the invention;
FIG. 2 is a schematic diagram of a microfluidic chip according to an exemplary embodiment of the present invention;
FIG. 3 is an optical photograph of a water-in-oil reaction droplet generated in a microfluidic chip according to an exemplary embodiment of the present invention;
FIG. 4 shows the results of the activity measurements of a cell sample according to an embodiment of the present invention;
FIG. 5 is an optical photograph of a water-in-oil reaction droplet generated in an embodiment of the present invention;
FIG. 6 is a Labchip detection profile of a cDNA pre-amplification product according to an embodiment of the present invention;
FIG. 7 is a Labchip detection profile of a sequencing library according to an embodiment of the present invention;
FIGS. 8A-8C show the nGene, nUMI, percent. Mito distribution of single cells, respectively, in an embodiment of the invention;
FIG. 9 is a diagram of cell clustering in accordance with one embodiment of the present invention;
FIG. 10 shows the result of sequencing quality assessment of read1 in accordance with an embodiment of the present invention;
FIG. 11 shows the result of sequencing quality assessment of read2 in an embodiment of the present invention.
Detailed Description
One aspect of the embodiments of the present invention provides a method for constructing a high-throughput single-cell transcriptome sequencing library, comprising:
capturing and packaging single cells by using a microfluidic chip so as to generate water-in-oil reaction droplets containing single cells;
cracking and reverse transcribing the cells in the water-in-oil reaction liquid drop to obtain a reverse transcription product;
performing demulsification treatment on the water-in-oil reaction liquid drop, and performing pre-amplification treatment on the reverse transcription product to obtain a pre-amplification product;
and (3) carrying out fragmentation treatment on the pre-amplification product, and then adding adapters at two ends of the obtained fragmentation product to generate a sequencing library.
In some embodiments, the construction method specifically comprises:
providing a micro-fluidic chip which comprises a cell micro-channel, a cell isolation medium micro-channel, a cell label micro-channel and a single-cell sample collecting port;
respectively injecting the cell suspension and the cell label suspension into the microfluidic chip, and mixing the cell suspension and the cell label suspension to form cell carrier liquid after the cell suspension and the cell label suspension respectively flow through the cell microchannel and the cell label microchannel;
injecting a cell isolation medium into the microfluidic chip, enabling the cell isolation medium to be in contact with the cell carrier liquid when flowing in the cell isolation medium microchannel, and shearing and wrapping the cell carrier liquid to form a water-in-oil reaction droplet containing a single cell and a single cell label;
and collecting the water-in-oil reaction droplets from the single cell sample collection port.
In some embodiments, the microfluidic chip comprises a cell microchannel, a cell isolation medium microchannel, a cell label microchannel and a single-cell sample collection port, the cell micro-flow channel is provided with a cell suspension inlet and a single cell suspension outlet, the cell isolation medium micro-flow channel is provided with a cell label suspension inlet and a cell label suspension outlet, and the single-cell suspension outlet is intersected with the cell label suspension outlet, so that the single-cell suspension output by the cell micro-channel can be mixed with the cell label suspension output by the cell label micro-channel to form cell carrier liquid, the flow path of the cell carrier liquid is intersected with the cell isolation medium micro-channel, so that the cell isolation medium flowing in the cell isolation medium micro-channel can be cut and wrapped with the cell carrier liquid, thereby forming a water-in-oil reaction droplet comprising a single cell which is output by the single cell sample collection port.
Of course, a transition section for the cell carrier liquid to flow may also be provided between the intersection of the single-cell suspension outlet and the cell label suspension outlet and the cell isolation medium micro-channel, which may be named as a cell carrier liquid micro-channel (see reference numeral 13 in fig. 2), and the cell carrier liquid micro-channel intersects with the cell isolation medium micro-channel.
Furthermore, the tail region of the cell micro-channel is set as a single-cell channel, the width of the single-cell channel is equal to or slightly larger than the diameter of a single cell, the outlet of the single-cell channel intersects with the outlet of the cell label micro-channel, so that the single-cell suspension output from the cell micro-channel and the cell label suspension output from the cell label micro-channel are mixed to form a cell carrier liquid, the continuous flow path of the cell carrier liquid intersects with the cell isolation medium micro-channel, so that the cell isolation medium flowing through the cell isolation medium micro-channel can shear the continuous cell carrier liquid into discrete droplet-shaped cell liquid phase and enable each cell liquid phase to contain a single cell and a single cell label, and meanwhile, the cell isolation medium is used as an oil phase to wrap the cell liquid phase, so that the water-in-oil reaction liquid droplet is formed.
Further, the water-in-oil reaction droplets act as water-in-oil microreactors, which can be pico-liter in size.
Furthermore, the microfluidic chip also comprises a cell suspension sample adding cup, a cell isolation medium sample adding cup and a cell label sample adding cup which are respectively communicated with the cell micro-channel, the cell isolation medium micro-channel and the cell label micro-channel.
In some embodiments, a negative pressure motive force generation device is disposed at the single-cell sample collection port. The negative pressure power generation device can adopt an air pump and the like, and can generate negative pressure in the microfluidic chip so as to drive fluid in each micro-channel to flow. For example, air can be pumped out by an air pump at the single cell sample collecting port, and negative pressure of-4K to-10K Pa can be applied to the whole microfluidic chip. Through adopting this kind of negative pressure mode, and set up the chip and be 3 passageways, need not drive with power, compare malleation drive among the prior art, multichannel (more than 4 passageways), it is more convenient to operate, and the time also shortens greatly, can do trace sample, and the flexibility is higher, can do 1 to 8 samples simultaneously.
Further, the structure of the microfluidic chip in the foregoing embodiment can be seen from fig. 2, and includes a cell suspension sample cup 1, a cell label sample cup 2, and a cell isolation medium sample cup 4, where the cell suspension sample cup 1, the cell label sample cup 2, and the cell isolation medium sample cup 4 are respectively communicated with a cell micro flow channel 11, a cell label micro flow channel 12, and a cell isolation medium micro flow channel 14, and the microfluidic chip is further provided with a single-cell sample collection port 3. Wherein, the cell micro-flow channel 11 and the cell label micro-flow channel 12 are crossed with each other and then crossed with the cell isolation medium micro-flow channel 14, and further communicated with the single cell sample collection port 3.
In the above embodiment of the present invention, the cell microchannel of the microfluidic chip is a flow channel for forming a cell suspension of one component of the cell liquid phase, the cell label channel is a flow channel for forming a cell label suspension of another component of the cell liquid phase, the cell isolation medium microchannel is a flow channel for a component of the oil phase, all the components flow at a certain speed along with the flow channel thereof under the condition that pressure is applied to the chip, and the cell suspension and the cell label suspension are mixed to form cell carrier liquid which is cut by a cell isolation medium as an oil phase to form physical isolation, through controlling the pressure and flow resistance design, the cell isolation medium cuts single cells and cell labels, the separation of the single cells and gel microbeads is realized, and each water-in-oil reaction liquid drop is ensured to be used as a micro-reaction system and comprises one cell and one cell label.
In some embodiments, the oil phase comprises an oil and a cell lysis reagent and the cellular liquid phase comprises an RNA reverse transcription module.
In some embodiments, the volume ratio of oil to cell lysis reagent is 100: 1-500: 1, for example, may be 100: 1. 200: 1. 300, and (2) 300: 1. 500: 1, etc., but are not limited thereto.
In some embodiments, the cell tag comprises a tagged polyT primer and a deformable microbead, the tagged polyT primer is attached to the deformable microbead, and the tagged polyT primer is capable of detaching from the deformable microbead by physical and/or chemical action.
Wherein, RNA can be better captured through a polyT sequence in the polyT primer.
Further, the physical and chemical actions include various physical and chemical actions known to those skilled in the art, such as ultraviolet light irradiation or specific enzyme digestion, etc., and are not limited thereto. For example, the polyT primer may be labeled as an oligonucleotide chain that is uv-sensitive, light-sensitive, or may be specifically cleaved, and is not limited thereto. According to the invention, mRNA free in a water-in-oil micro-reaction system can be three-dimensionally captured under the dissociation of the sequence with the label on the bead through a physical or chemical mode, and the efficiency is higher compared with the mode of directly capturing the mRNA by using the sequence on the bead. In the invention, ultraviolet illumination and other modes are preferably adopted to separate the labeled polyT primer from the deformable microbeads, which is not only very convenient, but also can not introduce other chemical substances into a micro-reaction system, thereby avoiding potential pollution risks.
Further, the label includes a barcode for identifying the cell. The barcode may have a length of 4 to 30nt, but is not limited to a base sequence and a certain sequence combination within this length range.
Further, the barcode may comprise 3 constant base sequences but is not limited to 3.
Further, the barcode may comprise 3nt of riboG bases, but is not limited to 3 nt.
Further, the total length of the tagged polyT primer may vary from 50nt to 200nt, and is not limited thereto.
In some embodiments, the tagged polyT primer may be chemically and/or physically attached to the deformable microbead. For example, the polyT primer and the microbead may be linked by means of a covalent bond, chemical polymerization, antigen-antibody binding, enzyme-catalyzed linking reaction, and the like, without being limited thereto.
In some embodiments, the deformable beads may be of an organic material or an inorganic-organic composite material, and may be, for example, polyacrylamide gel beads, agarose coated magnetic beads, silica beads, inert material-made beads, and the like, without being limited thereto. Preferably, the deformable beads can be gel beads, which is beneficial to further improving the efficiency of the microfluidic chip for carrying out water-in-oil reaction droplet packaging, and greatly improving the cell flux in cooperation with the microfluidic chip. The mechanism may be as follows: due to the adoption of the deformable beads, beads with molecular labels can be coated in each water-in-oil reaction droplet, the single coating rate can reach 100%, and if the beads are replaced by the hard beads, the droplets are coated according to the Poisson distribution rule, so that the actual coating efficiency is far lower than that of the deformable beads. Further, in the present invention, it is preferable to use porous polyacrylamide beads, which carry far more primers than other beads because their specific surface area is much larger than that of other beads, for example, hard beads such as resin beads or magnetic beads. When the polyacrylamide microbeads are synthesized, the concentration of the used acrylamide monomer can be 1% -10%.
In some embodiments, the diameter of the beads may be from 10 μ M to 200 μ M.
In the foregoing embodiment of the present invention, with the microfluidic chip, under the condition of the same concentration of cells, the single-port double-encapsulation rate is 1/2 with two ports, different cell sizes can be encapsulated by adjusting the pressure (for example, using the pressure generated by the negative pressure power generation device as the power source), especially when the negative pressure power generation device is used as the power source, the encapsulation of cells can be rapidly and efficiently completed, and when the gel beads are used to form cell labels, the flow rate of the suspension has impact force, and is controllable, so that the encapsulation rate of 90% or more can be achieved.
And, in the foregoing embodiments of the present invention, a water-in-oil microreactor with a pico-upgrade can be realized by a microfluidic chip technology, and compared with the prior art in which separation is performed in a 96-well plate, a larger number of cells can be detected under the condition of the same number of reverse transcription components, so that the detection cost of a single cell is greatly reduced, and separation and detection of up to 17000 cells can also be realized by increasing a molecular tag loaded on a gel bead.
In some embodiments, the water-in-oil reaction droplet further comprises a cell lysis reagent comprising a cell lytic enzyme or other substance capable of promoting cell lysis and an RNA reverse transcriptase reagent comprising a reverse transcriptase primer, an RNA reverse transcriptase inhibitor, and the like.
The invention adopts a template conversion mode to carry out reverse transcription reaction in water-in-oil reaction liquid drops, and simultaneously combines a transposase library building mode outside the liquid drops, the time of the whole process from reverse transcription to library building is less than 8 hours, and in contrast, the time of an in vitro transcription mode adopted in the prior art is generally more than 30 hours. In addition, the reverse transcription reaction mode adopted by the invention can avoid cross contamination caused by reverse transcription outside the liquid drop, effectively reduce double-package rate and reduce false positive results. And, it is also beneficial to simplify the subsequent library building operation, for example, there is no need to adopt a terminal-plus-a library building method which is complicated and time-consuming in operation.
In some embodiments, the construction method further comprises:
after demulsification treatment is carried out on the water-in-oil reaction liquid drop, purification and pre-amplification treatment are carried out on the reverse transcription product in sequence to obtain a pre-amplification product;
after purifying the pre-amplification product, fragmenting the pre-amplification product by using transposase, and after the fragmentation reaction is ended, purifying the obtained fragmentation product;
and adding linkers at two ends of the purified fragmentation product to generate a sequencing library, and purifying the sequencing library.
The demulsification treatment preferably adopts physical demulsification modes such as ultrasound and the like, so that the influence of chemical components such as PFO (Perfluorooctane sulfonate) existing in the chemical demulsification modes on subsequent reactions is avoided.
In some embodiments, the construction method may further include a step of pre-treating the "sample to be tested" or the "sample to be tested". However, with the method provided by the embodiment of the present invention, the requirement for pre-treatment is low, for example, preliminary enrichment can be performed according to the physical or biological characteristics of the cells, and the obtained sample can be used in the subsequent steps.
In this specification, a "test sample" or "test sample" may be derived from an individual (e.g., human blood, biological tissue, etc.) or may be derived from another source, such as some processed or unprocessed laboratory material. In addition, in the present specification, the detection of a "sample" or "specimen" is not only related to a diagnostic purpose, but may also be related to other non-diagnostic purposes.
In another aspect of the embodiments of the present invention, there is provided a kit for constructing a high-throughput single-cell transcriptome sequencing library, comprising:
a microfluidic chip for capturing at least single cells and packaging to generate a water-in-oil reaction droplet, the water-in-oil reaction droplet comprising an oil phase and a cellular liquid phase encapsulated by the oil phase, and the water-in-oil reaction droplet comprising single cells, a single cell tag, a cell lysis reagent, and an RNA reverse transcription reagent;
an oil for forming the oil phase;
a cell lysis reagent;
an RNA reverse transcription reagent;
a cell tag comprising a deformable microbead and a tagged polyT primer attached to the deformable microbead, the tagged polyT primer capable of detaching from the deformable microbead under physical and/or chemical action; and
cDNA pre-amplification reagents, transposition fragmentation reagents and reagents required to attach linkers at both ends of the fragmentation product.
In the kit provided in this embodiment, the structure and the working principle of the microfluidic chip are as described above, and are not described herein again.
In some embodiments, the oil phase comprises an oil and a cell lysis reagent. Wherein the volume ratio of the oil to the cell lysis reagent is 100: 1-500: 1, for example, may be 100: 1. 200: 1. 300, and (2) 300: 1. 500: 1, etc., but are not limited thereto.
In some embodiments, the cell fluid phase comprises single cells, a single cell tag, and a RNA reverse transcription reagent.
In the kit provided in this embodiment, the composition of the cell label may be as described above, and is not described herein again.
In some embodiments, the kit further comprises: reagents required for purification of any one or more of the reverse transcription product, the cDNA pre-amplification product, the fragmentation product, and the sequencing library, such as magnetic beads, etc., are not limited thereto.
In the previous embodiments of the present invention, the cell lysis reagent may be selected from the types well known to those skilled in the art, and may include any protease and protein denaturing reagent suitable for cell lysis, lysis buffer system, etc. well known to those skilled in the art.
In the foregoing embodiment of the present invention, the RNA reverse transcription reagent may comprise an RNA reverse transcription primer and RNA reverse transcriptase, an RNA reverse transcriptase inhibitor, an RNA reverse transcription buffer, and the like, which are well known to those skilled in the art.
For example, the reverse transcriptase can include M-MLV reverse transcriptase, which is an RNA template dependent DNA polymerase.
For example, the reverse transcription buffer may comprise Tris-HCl, KCl, MgCl as main component2、DTT、Mn2+Ions and water, wherein the content of each component can be as follows: concentration range of Tris-HCl 50-500mM (pH 7.0-9.0), concentration range of KCl 50-500mM, MgCl2The concentration range of (A) is 10mM-25mM, and the concentration range of DTT is 10mM-100 mM.
In the foregoing embodiments of the invention, the cDNA pre-amplification reagents may comprise cDNA pre-amplification primers and DNA polymerase and amplification buffer well known to those skilled in the art. For example, the amplification buffer, i.e., the cDNA pre-amplification reaction solution, may comprise, as essential components: KCl, NH4Cl、NaCl、Tris、MgCl2Betaine, DMSO, water, and the like. For example, the DNA polymerase, i.e., cDNA preamplifying enzyme, may be selected from Taq DNA polymerase, Hot Start Taq polymerase, high Fidelity enzyme, and the like.
In the preceding embodiments of the invention, the DNA polymerase may be any thermostable DNA polymerase known to those skilled in the art, for example: LA-Taq, rTaq, Phusion, Deep Vent (exo-), Gold 360, Platinum Taq, KAPA 2G Robust, and the like, without being limited thereto.
In the foregoing embodiments of the present invention, the transposition fragmenting reagent may comprise transposase, transposase reaction buffer, transposition reaction stop solution, and the like, which are well known to those skilled in the art. For example, the transposase may be Tn5 or the like, but is not limited thereto.
Yet another aspect of an embodiment of the present invention provides a method for high throughput single cell transcriptome sequencing, comprising: a single cell transcriptome sequencing library was constructed using any of the methods described previously, followed by sequencing analysis.
Further, the sequencing analysis may also be performed in a manner well known to those skilled in the art. Reference may be made, for example, to FIG. 4, which may include a base analysis, a standard analysis, and a high-level analysis.
According to the embodiment of the invention, the gel beads are adopted to construct the cell labels, reverse transcription is carried out in the water-in-oil reaction liquid drops, meanwhile, an air pump and the like are used as a power source to drive the generation of the water-in-oil reaction liquid drops, a plurality of samples can be simultaneously made, the capture efficiency of cells is effectively improved, the mutual pollution of the beads after the liquid drops are demulsified is reduced, the ratio of effective data is improved, the cost is reduced, the use mode is more flexible, the overall operation time is shortened to 7 hours from 30 hours compared with the existing indep and hydropseq platforms, and the operation is easier. In addition, the primers on the microbeads are released by ultraviolet or other chemical means, mRNA which is free in the liquid drops can be captured three-dimensionally, and the efficiency of capturing the mRNA is higher than that of capturing the mRNA by directly using the primers connected to the microbeads in a conventional mode. More specifically, the invention has high cell capture efficiency and high flux of 10 by reverse transcription in water-in-oil reaction liquid drops5(ii) individual cells; for another example, the invention adopts gel beads carrying polyT primers in water-in-oil reaction droplets, releases the polyT primers through ultraviolet irradiation to capture mRNA and carry out reverse transcription to form cDNA, and simultaneously utilizes an air pump to drive generation of the water-in-oil reaction droplets, so that 1-8 samples can be simultaneously prepared, and the flexibility is high. In addition, the invention also obviously improves the ratio of effective data, and has the advantages of low cross contamination, low double-package rate, low cost (less than one third of the sequencing mode based on platforms such as droseq, 10X and the like), and sequencing result accuracyHigh and the like, and has wide application prospect in a plurality of fields. In addition, the sequencing library construction process and the sequencing process can be fully automated, electric drive is not needed, and the corresponding kit is very convenient to carry.
The invention is further illustrated by the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Unless otherwise specified, various reagents used in the following examples are well known to those skilled in the art and available from commercial sources and the like. However, the experimental methods in the following examples, in which specific conditions are not specified, are generally performed under conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or under the conditions recommended by the manufacturers.
The following examples use a kit for constructing a human single-cell BCR sequencing library, which contains reagents and consumables required for constructing a single-cell immune repertoire library, such as: the microfluidic chip has the functions of single cell separation and water-in-oil reaction droplet generation; oil required for generating the reaction droplets; hydrogel microbeads loaded with labeled polyT primers; reverse transcription primer (see SEQ ID NO: 1); RNA reverse transcriptase; RNA reverse transcription buffer solution; nuclease-free water; taq polymerase reaction solution; cDNA amplification reaction buffer solution; cDNA amplification primers (see SEQ ID NO: 2); a cDNA amplification enzyme; transposase reaction buffer; a transposase; a transposition reaction stop solution; magnetic beads for double-stranded DNA purification; adaptors for library amplification, and the like, without limitation. The aforementioned reagents are commercially available unless otherwise specified. For example, the purified magnetic beads used in the following examples may preferably be Ampure XP beads from Beckman, SPRI beads from Beckman, and the like, and are not limited thereto.
Referring to FIG. 1, the sequencing library construction and sequencing process of the following example may include the following steps:
(1) digesting the tissue or the cell to obtain a single cell suspension;
(2) using hydrogel microbeads carrying a labeled polyT primer as a cell label and a droplet microfluidic technology (based on the implementation of a microfluidic chip shown in FIG. 2), respectively wrapping a single cell suspension and the cell label in a water-in-oil reaction droplet (hereinafter referred to as a "droplet") and treating the droplet (for example, UV irradiation), so that the labeled polyT primer on the hydrogel microbeads is released to allow the labeled polyT primer to capture mRNA through polyT;
(3) incubating the drop at a temperature (e.g., 55 ℃) for a time (e.g., 1.5 hours) to reverse transcribe the mRNA to cDNA;
(4) demulsifying, namely extracting cDNA by using magnetic beads and the like;
(5) fragmenting the extracted cDNA by means of ultrasound, PCR and the like, and adding sequencing connectors on two sides;
(6) performing second-generation sequencing on the built library, wherein the sequencing scheme can be PE150 or other schemes;
(7) the information data is interpreted to determine cell subpopulations.
The specific implementation of this example is described in more detail below.
1. Preparation of the experiment
1.1 oil phase (i.e.cell isolation Medium as described above, see Table 1 for details of composition)
TABLE 1 oil phase Components
| Composition (I) | Volume (μ L) |
| Oil (NO.036) | 800 |
| Cell lysis reagent (e.g., NP40) | 200 |
1.2 cell phase (i.e., cell suspension) (see Table 2 for details)
TABLE 2 cellular phase Components
Note: after the cell phase cells are detected by using a fluorescence cell analyzer for concentration and activity, the required volume is calculated according to the experiment requirement.
1.3 beads phase (i.e., cell-tag suspension) (see Table 3 for details)
TABLE 3 aqueous phase composition
| Composition (I) | Volume (μ L) |
| |
10 |
Note: after the Beads are detected by a fluorescence cell analyzer for concentration and diameter, the required volume is calculated according to the experiment requirement.
The gel beads constituting the cell tag may be selected from polyacrylamide gel beads, agarose gel beads, etc. having a diameter of 10 μ M to 200 μ M, and the ployT primer attached to the gel beads is an ultraviolet-sensitive oligonucleotide strand, which may have a length of 50nt to 200nt, including a barcode having a length of about 8nt, which may include 3 or more constant base sequences, and which may include 3nt or more riboG bases. For example, the cell signature can be expressed as: microbead-5 '-CGATGACGCTACACGACGACGCTTCCGATCTACjjjjjgtGATTGTGACTGTGACjjjjjjCGACTCACACACACACACACACAGCcjjjjjjjjJNNNNNNNNNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3'. Wherein, the sequence shown by j and N is a tag sequence, which can be a random sequence.
2. Experimental operation and result display
2.1 cell preparation
2.1.1
And taking a proper amount of PBS solution according to the requirement of the number of processed samples, and putting the PBS solution into a water bath kettle for preheating half an hour in advance.
2.1.2
Taking a fresh lung cancer sample of 1cm3Digested to single cells with enzyme, suspended in pre-warmed PBS. The cell activity (87%) was measured as shown in FIG. 4. Wherein green indicates viable cells, stained with AO; red indicates dead cells, stained with PI.
2.2 Single cell encapsulation
2.2.1 machine-generated droplets
The single cell suspension, the cell label suspension and the oil phase are respectively added into the microfluidic chip shown in figure 2. The volume was pulled from 20ml to 30ml by air pump. The process in which water-in-oil reaction droplets are generated is shown in FIG. 3. The morphology of the resulting droplets is shown in FIG. 5.
2.2.2 cDNA reverse transcription
The temperature control setting at the collection tube was as follows: RT reaction (reaction temperature: 42 ℃ reaction time: 90min) was carried out to obtain an RT product (i.e., cDNA).
2.2.3 demulsification
Adding 3ml PFO into the collecting pipe, blowing and beating for 15 times by using a gun head, centrifuging (800g, 10min, 4 ℃), and taking supernatant containing RT product, namely cDNA;
2.3 RT product purification
2.3.1 Advance
SILANE viral NA kit is balanced for more than 30min at room temperature;
2.3.2
SILANE viral NA under the condition that the kit is used for the first time, according to the requirements of a reagent specification, adding isopropanol into Washing Buffer 1, and adding absolute ethyl alcohol into Washing Buffer 2;
2.3.3 transfer RT product to a new 1.5mL centrifuge tube and fill the sample volume to 250. mu.L;
2.3.4 adding 300. mu.L of lysine/binding Buffer to the sample, mixing by reversing the top and the bottom, and standing at room temperature for 5 min;
2.3.5 Add 150. mu.L of Isopropanol to the reaction mixture from the previous step, add 50. mu.L of the mixture and mix
MyOneTMSILANE, mixing by turning upside down, and incubating for 10min by shaking;
2.3.6 centrifuging the centrifuge tube instantly, collecting the sample solution on the tube cover, placing the centrifuge tube on a magnetic frame for 2min until the solution is clear, and removing the supernatant;
2.3.7 taking down the centrifugal tube from the magnetic frame, adding 850 mu L Washing Buffer 2 into the centrifugal tube, blowing, sucking and uniformly mixing for 4-5 times, placing the centrifugal tube on the magnetic frame for 2min until the solution is clarified, and sucking and removing the supernatant;
2.3.8 repeat step 2.3.7 once;
2.3.9 taking the centrifuge tube off the magnetic frame, adding 450 μ L Washing Buffer 1, blowing and sucking, mixing 4-5 times, and transferring the solution to a new 1.5mL centrifuge tube; placing the centrifuge tube on a magnetic frame for 1min until the solution is clear, and sucking and removing the supernatant;
2.3.10 repeat step 2.3.9 once without changing a new 1.5mL centrifuge tube;
2.3.11 air drying the magnetic beads at room temperature for 10-15 min;
2.3.12 mu.L RNase Free H was added2O, incubating at 70 ℃ for 3min, placing the centrifuge tube on a magnetic frame for 2min until the solution is clear, sucking 25 mu L of supernatant, and transferring the supernatant into a clean 0.2mL PCR tube;
2.3.13 Qubit quantification was performed on the purified reverse transcription product and the concentration was recorded.
2.4 cDNA Pre-amplification
2.4.1 preparation of the reaction System and setting of the reaction program
1) And (3) PCR reaction system:
| reagent | Volume of |
| 2×KAPA HIFI HotStart Ready Mix | 25μL |
| Purified reverse transcription product | 24μL |
| Reverse transcription primer (20uM) | 1μL |
2) PCR reaction procedure:
the products were purified using 0.6 XKAPA Pure beads for 2100 detection, with a main peak around 1.2-1.7K, as shown in FIG. 6.
2.4 library construction
2.4.1 fragmentation treatment
Preparing reaction system and setting reaction program
1) Reaction system:
2) reaction procedure:
| temperature of | Time | |
| 75℃ | Hot lid | |
| 55 | 5min | |
| 12℃ | Hold |
2.4.2 termination of fragmentation
After the reaction was complete, the PCR tube was removed immediately, 10. mu.L of 6 × Termination Buffer was added, mixed by pipetting, and incubated at room temperature for 5 min.
2.4.3 purification of the fragmentation product ((1.0 XKAPA Pure beads purification)
2.4.4 PCR enrichment with adaptor
Preparing reaction system and setting reaction program
1) Reaction conditions are as follows:
| reagent | Volume/. |
| 2×PCR Mix | 25μL |
| Fragmentation products | 15μL |
| Truseq-adapter(10uM) | 5μL |
| N7(10uM) | 5μL |
| Total | 50μL |
2) Reaction procedure:
2.4.5 with 0.6 × +0.15 × KAPA Pure beads purification library, using 2100 detection, wherein the main peak is 464bp, see figure 7.
3. Sequencing analysis
The sequencing process adopts a PE150 sequencing scheme, can be carried out on Illumina Nova Seq, Illumina Hiseq, Illumina Nextseq 500, Illumina Miseq and other platforms, and corresponding operation methods and experimental conditions are well known to those skilled in the art.
For this example, FIG. 8A shows the number of gene tests per single cell. FIG. 8B shows the number of captured cellular transcripts. FIG. 8C shows the number of mitochondrial genes detected per cell, indicating the activity state of the cell. FIG. 9 shows cell clustering in which different subpopulations are separated by dimension reduction analysis based on the difference in gene expression level of each cell, and each color indicates a different population. The results of the quality evaluation of the sequencing data of this example are shown in FIGS. 10 to 11.
By using the cell sample same as the cell sample in the embodiment of the invention and comparing the obtained sequencing result with the sequencing result of the embodiment of the invention based on the 10X Genomics platform according to the manner known by the technicians in the field, the invention can find that the obtained sequencing result is accurate, the sensitivity is high, and the efficiency, the cost and the like are far superior to the sequencing scheme based on the 10X Genomics platform.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
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Claims (19)
1. A method for constructing a high-throughput single-cell transcriptome sequencing library, which is characterized by comprising the following steps:
capturing and packaging single cells by using a microfluidic chip so as to generate water-in-oil reaction droplets containing single cells;
cracking and reverse transcribing the cells in the water-in-oil reaction liquid drop to obtain a reverse transcription product;
performing demulsification treatment on the water-in-oil reaction liquid drop, and performing pre-amplification treatment on the reverse transcription product to obtain a pre-amplification product;
and (3) carrying out fragmentation treatment on the pre-amplification product, and then adding adapters at two ends of the obtained fragmentation product to generate a sequencing library.
2. The construction method according to claim 1, characterized by specifically comprising:
providing a micro-fluidic chip which comprises a cell micro-channel, a cell isolation medium micro-channel, a cell label micro-channel and a single-cell sample collecting port;
respectively injecting the cell suspension and the cell label suspension into the microfluidic chip, and mixing the cell suspension and the cell label suspension to form cell carrier liquid after the cell suspension and the cell label suspension respectively flow through the cell microchannel and the cell label microchannel;
injecting a cell isolation medium into the microfluidic chip, enabling the cell isolation medium to be in contact with the cell carrier liquid when flowing in the cell isolation medium microchannel, and shearing and wrapping the cell carrier liquid to form a water-in-oil reaction droplet containing a single cell and a single cell label;
and collecting the water-in-oil reaction droplets from the single cell sample collection port.
3. The construction method according to claim 2, wherein: the cell micro-channel is provided with a cell suspension inlet and a single-cell suspension outlet, the cell isolation medium micro-channel is provided with a cell label suspension inlet and a cell label suspension outlet, the single-cell suspension outlet is intersected with the cell label suspension outlet, so that the single-cell suspension output by the cell micro-channel can be mixed with the cell label suspension output by the cell label micro-channel to form cell carrier liquid, the flow path of the cell carrier liquid is intersected with the cell isolation medium micro-channel, the cell isolation medium flowing in the cell isolation medium micro-channel can be cut and wraps the cell carrier liquid, water-in-oil reaction liquid drops containing single cells are formed, and the water-in-oil reaction liquid drops containing the single cells are output from the single-cell sample collection port.
4. The construction method according to claim 3, wherein: the tail area of the cell micro-channel is set as a single-cell channel, the width of the single-cell channel is equal to or slightly larger than the diameter of a single cell, the outlet of the single-cell channel is intersected with the outlet of the cell label micro-channel, so that a single-cell suspension output from the cell micro-channel and a cell label suspension output from the cell label micro-channel are mixed to form a cell carrier liquid, the continuous flow path of the cell carrier liquid is intersected with the cell isolation medium micro-channel, the cell isolation medium flowing through the cell isolation medium micro-channel can shear the continuous cell carrier liquid into discrete droplet-shaped cell liquid phases and enable each cell liquid phase to contain a single cell and a single cell label, and meanwhile, the cell isolation medium is used as an oil phase to wrap the cell liquid phase, so that the water-in-oil reaction liquid droplet.
5. The construction method according to claim 3 or 4, characterized in that: and a negative pressure power generation device arranged at the single cell sample collection port is utilized to generate negative pressure in the microfluidic chip, so that the fluid in each micro-channel is driven to flow.
6. The construction method according to any one of claims 1 to 4, characterized in that: the water-in-oil reaction droplet also contains a cell lysis reagent and an RNA reverse transcription reagent.
7. The construction method according to any one of claims 2 to 4, wherein: the cell tag comprises a tagged polyT primer and a deformable microbead, wherein the tagged polyT primer is connected to the deformable microbead, and the tagged polyT primer can be separated from the deformable microbead under physical action and/or chemical action; preferably, the deformable beads comprise porous polyacrylamide beads; preferably, the diameter of the deformable beads is 10 μ M to 200 μ M.
8. The construction method according to claim 7, characterized by further comprising: the water-in-oil reaction droplet is irradiated with ultraviolet light, thereby releasing the tagged polyT primer from the deformable beads therein.
9. The building method according to claim 1, characterized by further comprising:
after demulsification treatment is carried out on the water-in-oil reaction liquid drop, purification and pre-amplification treatment are carried out on the reverse transcription product in sequence to obtain a pre-amplification product;
after purifying the pre-amplification product, fragmenting the pre-amplification product by using transposase, and after the fragmentation reaction is ended, purifying the obtained fragmentation product;
and adding linkers at two ends of the purified fragmentation product to generate a sequencing library, and purifying the sequencing library.
10. The construction method according to claim 1 or 9, characterized by comprising: demulsifying the water-in-oil reaction liquid drop by adopting a physical mode; preferably, the physical means comprises sonication means.
11. A high throughput single cell transcriptome sequencing method, comprising: constructing a single cell transcriptome sequencing library using the method of any one of claims 1 to 10, followed by sequencing analysis.
12. A kit for constructing a high throughput single cell transcriptome sequencing library, comprising:
a microfluidic chip for capturing at least single cells and packaging to generate a water-in-oil reaction droplet, the water-in-oil reaction droplet comprising an oil phase and a cellular liquid phase encapsulated by the oil phase, and the water-in-oil reaction droplet comprising single cells, a single cell tag, a cell lysis reagent, and an RNA reverse transcription reagent;
an oil for forming the oil phase;
a cell lysis reagent;
an RNA reverse transcription reagent;
a cell tag comprising a deformable microbead and a tagged polyT primer attached to the deformable microbead, the tagged polyT primer capable of detaching from the deformable microbead under physical and/or chemical action; and
cDNA pre-amplification reagents, transposition fragmentation reagents and reagents required to attach linkers at both ends of the fragmentation product.
13. The kit of claim 12, wherein: the micro-fluidic chip comprises a cell micro-channel, a cell isolation medium micro-channel, a cell label micro-channel and a single-cell sample collecting port, the cell micro-flow channel is provided with a cell suspension inlet and a single cell suspension outlet, the cell isolation medium micro-flow channel is provided with a cell label suspension inlet and a cell label suspension outlet, and the single-cell suspension outlet is intersected with the cell label suspension outlet, so that the single-cell suspension output by the cell micro-channel can be mixed with the cell label suspension output by the cell label micro-channel to form cell carrier liquid, the flow path of the cell carrier liquid is intersected with the cell isolation medium micro-channel, so that the cell isolation medium flowing in the cell isolation medium micro-channel can be cut and wrapped with the cell carrier liquid, thereby forming a water-in-oil reaction droplet comprising a single cell which is output by the single cell sample collection port.
14. The kit of claim 12, wherein: the tail area of the cell micro-channel is set as a single-cell channel, the width of the single-cell channel is equal to or slightly larger than the diameter of a single cell, the outlet of the single-cell channel is intersected with the outlet of the cell label micro-channel, so that a single-cell suspension output from the cell micro-channel and a cell label suspension output from the cell label micro-channel are mixed to form a cell carrier liquid, the continuous flow path of the cell carrier liquid is intersected with the cell isolation medium micro-channel, the cell isolation medium flowing through the cell isolation medium micro-channel can shear the continuous cell carrier liquid into discrete droplet-shaped cell liquid phases and enable each cell liquid phase to contain a single cell and a single cell label, and meanwhile, the cell isolation medium is used as an oil phase to wrap the cell liquid phase, so that the water-in-oil reaction liquid droplet.
15. The kit according to claim 13 or 14, characterized in that: the microfluidic chip also comprises a cell suspension sample adding cup, a cell isolation medium sample adding cup and a cell label sample adding cup which are respectively communicated with the cell micro-channel, the cell isolation medium micro-channel and the cell label micro-channel.
16. The kit according to claim 13 or 14, characterized in that: and a negative pressure power generation device is arranged at the single cell sample collecting opening.
17. The kit of claim 12, wherein: the oil phase comprises oil and a cell lysis reagent, and the cell liquid phase comprises single cells, a single cell tag and an RNA reverse transcription component; preferably, the volume ratio of the oil to the cell lysis reagent is 100: 1-500: 1.
18. the kit of claim 12, wherein: the physical effect comprises ultraviolet light irradiation; alternatively, the chemical action comprises specific enzymatic cleavage; and/or, the deformable beads comprise porous polyacrylamide beads; and/or the diameter of the deformable microbead is 10-200 μ M.
19. The kit of claim 12, further comprising: magnetic beads required for purification of any one or more of reverse transcription products, cDNA pre-amplification products, fragmentation products, and sequencing libraries.
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| CN201911353094.XA CN113026110A (en) | 2019-12-25 | 2019-12-25 | High-throughput single-cell transcriptome sequencing method and kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115198001A (en) * | 2022-07-21 | 2022-10-18 | 北京寻因生物科技有限公司 | Single cell complete sequence transcriptome library construction method and application |
| CN115386624A (en) * | 2022-10-26 | 2022-11-25 | 北京寻因生物科技有限公司 | Single cell complete sequence marking method and application thereof |
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2019
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115198001A (en) * | 2022-07-21 | 2022-10-18 | 北京寻因生物科技有限公司 | Single cell complete sequence transcriptome library construction method and application |
| CN115198001B (en) * | 2022-07-21 | 2023-07-04 | 北京寻因生物科技有限公司 | Construction method and application of single-cell complete sequence transcriptome library |
| CN115386624A (en) * | 2022-10-26 | 2022-11-25 | 北京寻因生物科技有限公司 | Single cell complete sequence marking method and application thereof |
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