CN112795563A - Use and method of biotinylated transposomes for recovering CUT & Tag or ATAC-seq products - Google Patents
Use and method of biotinylated transposomes for recovering CUT & Tag or ATAC-seq products Download PDFInfo
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Abstract
The invention relates to the fields of molecular biology, genomics and biotechnology, in particular to application and a method of a biotinylated transposome in recovery of CUT & Tag or ATAC-seq products, wherein the biotinylated transposome is selected from a biotinylated transposome formed by a fusion protein carrying transposase and a biotinylated linker-terminal complex capable of combining with the transposase, and/or a biotinylated transposome formed by combining the transposase and the biotinylated linker-terminal complex; the fusion protein carrying transposase includes transposase and a protein having a function of binding to an Fc fragment of an antibody. Biotinylated transposomes can be used to recover the CUT & Tag or ATAC-seq products. The recovery method provided by the application is convenient and rapid, has higher product recovery efficiency, has no obvious substrate loss, and is particularly suitable for epigenetics and chromatin state research of a small number of cells.
Description
Technical Field
The invention relates to the fields of molecular biology, genomics and biotechnology, in particular to the use and method of biotinylated transposomes in the recovery of CUT & Tag or ATAC-seq products.
Background
Chromatin targeted Cleavage and labeling (CUT & Tag) is a new protein-Chromatin interaction research method that has been emerging in recent years, which combines the characteristics of transposition-Accessible Chromatin experiments (Assay for Targeting access-Chromatin with high-throughput sequencing, ATAC-seq) and Chromatin nuclease targeted Cleavage and release strategies (CUT & RUN), and utilizes the fusion protein of protein a/protein G fusion Tn5 to tether a transposable body in the region around a target antibody through protein a/G-antibody interaction, thereby realizing target-specific fragmentation reaction. Compared with the traditional ChIP-seq, the CUT & Tag has the advantages of relatively simple operation, no need of formaldehyde crosslinking, high signal-to-noise ratio, less required sequencing amount and suitability for application scenes with extremely low initial quantity and single cells. Transposomes used in CUT & Tag and ATAC-seq need to be inactivated by adding SDS, the SDS existing in the system can interfere the subsequent PCR reaction, and therefore DNA extraction or other methods for eliminating the interference of the SDS are needed. The existing recovery or subsequent treatment method of the CUT & Tag/ATAC-seq product mainly comprises a phenol chloroform extraction-ethanol precipitation method, a fragment-Separation (SPRI) magnetic bead recovery method, a centrifugal column recovery method and a direct PCR method, wherein the phenol chloroform extraction-ethanol precipitation method has the disadvantages of complicated operation, long time consumption and relatively low efficiency; the SPRI beads recovery method can lose fragments smaller than 150bp, thereby causing signal loss; the centrifugal column recovery method needs high-quality centrifugal columns, and increases the material cost; while the direct PCR method will lose more than 300bp fragments on part of the antibody, and the library construction effect is greatly influenced by the amplification enzyme and the extension time. The existing recovery/subsequent treatment methods of the CUT & Tag products have respective defects, which influence the wider application of the CUT & Tag technology, so that the establishment of a universal, convenient, fast and substrate-loss-free recovery method of the CUT & Tag/ATAC-seq products is very necessary.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide the use and methods of biotinylated transposomes for the recovery of CUT & Tag or ATAC-seq products, for solving the problems of the prior art.
To achieve the above and other related objects, the present invention provides the use of biotinylated transposomes selected from the group consisting of biotinylated transposomes formed from a fusion protein carrying a transposase and a biotinylated linker-terminal complex capable of binding to the transposase, and/or biotinylated transposomes formed from a transposase bound to a biotinylated linker-terminal complex, for recovering CUT & Tag or ATAC-seq products; the fusion Protein carrying the transposase also comprises a Protein with the function of binding the Fc segment of the antibody, and the Protein with the function of binding the Fc segment of the antibody is selected from more than two of Protein A, Protein G or Protein L.
The present invention also provides a method for recovering a fragmented nucleic acid product that binds to a target protein, the recovery method comprising the steps of:
1) selected from any one of:
1a) incubating the cell nucleus or permeabilized cell with an antibody and a biotinylated transposome; performing an enzymatic cleavage reaction on the biotinylated transposome guided by the antibody and generating a fragment containing the linker sequence;
1b) incubating the nucleus or permeabilized cell with a biotinylated transposome; directly carrying out enzyme digestion reaction through a biotinylated transposome and generating a fragment containing a linker sequence;
2) inactivating the biotinylated transposomes added in step 1) such that biotinylated fragments are released into the solution supernatant;
3) and then adding streptavidin magnetic beads, and recovering to obtain a fragmented nucleic acid product combined with the target protein.
As described above, the use and method of the biotinylated transposomes of the invention for recovering the CUT & Tag or ATAC-seq products has the following beneficial effects: according to the method, the joint sequence of the transfer base body in CUT & Tag or ATAC-seq is biotinylated, the generated biotinylated nucleic acid product can be combined by streptavidin magnetic beads, the streptavidin magnetic beads can be combined with all biotinylated DNA only within 20 minutes of room temperature incubation time after being recovered, and the NGS library can be directly prepared by PCR after washing. Through the detection of the recovery efficiency and the library distribution, the recovery method provided by the application is found to have higher product recovery efficiency while being convenient and quick, and has no obvious substrate loss caused by fragment preference. The operation difficulty is reduced, the recovery efficiency is improved, and the method is particularly suitable for a recovery scene of a small amount of cell trace DNA.
Drawings
FIG. 1 shows a schematic diagram of the CUT & Tag product recovery process.
FIG. 2 shows a comparison of the conventional pAG-Tn5 and biotinylated pAG-Tn5 labeling reactions.
FIG. 3 shows the nucleic acid-free band of the supernatant after incubation of streptavidin magnetic beads with biotinylated fragmentation products.
FIG. 4 shows that CUT & Tag for biotinylated pAG-Tn5 gave similar effects to normal transposomes.
FIG. 5 shows the fragment distribution of the CUT & Tag products obtained for different recovery methods.
FIG. 6 shows the recovery of the CUT & Tag product for SPRI beads and the distribution of the CUT & Tag product recovered from biotin adaptor-pAG-Tn5 (i.e., SA beads) on the HPRT1 gene, and the contribution of different size fragments in the CUT & Tag product recovered from biotin adaptor-pAG-Tn5 to the results.
FIG. 7 shows the distribution contribution of the CUT & Tag product on the KRAS gene recovered by SPRI beads, ethanol precipitation-phenol chloroform extraction (i.e., ET), direct PCR method (i.e., D-PCR) and biotin adaptor-pAG-Tn5 (i.e., SA beads) and the effect of the recovery data volume of different SPRI beads on the results.
FIG. 8 shows a comparison of the sizes of the four different recovery methods at the scale of the library generated.
FIG. 9 shows the fragment distribution plots of the ATAC-seq products obtained for the different recovery methods.
FIG. 10 is a graph showing the effect of ATAC-seq products obtained from different recovery methods on a representative section.
Detailed Description
The invention provides the use of biotinylated transposomes in the recovery of CUT & Tag or ATAC-seq products, said biotinylated transposomes being selected from the group consisting of biotinylated transposomes formed from a fusion protein carrying a transposase and a biotinylated linker-terminus complex capable of binding to the transposase, and/or biotinylated transposomes formed from a transposase bound to a biotinylated linker-terminus complex; the fusion Protein carrying the transposase also comprises a Protein with the function of binding the Fc segment of the antibody, and the Protein with the function of binding the Fc segment of the antibody is selected from more than two of Protein A, Protein G or Protein L.
The biotinylated transposome described above can be referred to as a biotinylated transposome formed by a fusion protein carrying a transposase and a biotinylated linker-terminus complex.
In one embodiment, the transposase-carrying fusion protein includes a transposase and a protein having a function of binding to an Fc fragment of an antibody.
The transposase is selected from the group consisting of Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn7, Tn8, Tn9, and Tn 10. Preferably, the transposase is selected from Tn 5.
Preferably, the function of binding to the Fc segment of the antibody refers to the function of binding to the Fc segment of an IgG molecule.
The Protein with the function of binding the Fc segment of the antibody is selected from one or more of staphylococcus aureus Protein A (Protein A), streptococcus G Protein (Protein G) or streptococcus L Protein (Protein L). In a preferred embodiment, two or more selected from Protein A, Protein G or Protein L. In a more preferred embodiment, the Protein having the function of binding to the Fc fragment of an antibody is selected from the group consisting of Protein A and Protein G.
In one embodiment, the fusion Protein carrying transposase is a Protein fused by Protein A, Protein G and Tn5, i.e., pAG-Tn 5. pAG-Tn5 is commercially available, for example, from CUTANA ™ pAG-Tn5 for ChIC/CUT & Tag (manufacturer: epicypher, cat. No. 15-1017). The use of pAG-Tn5 can improve the experimental efficiency of CUT & Tag and ATAC-seq, the yield of the obtained library is more than that of pA-Tn5 in the prior art, the restriction of antibody selection is removed, and the requirement of the experiment on a secondary antibody is reduced.
Further, the single-chain linker A and the single-chain linker B are linkers modified by biotin. Further, the 5' ends of the single-chain linker A and the single-chain linker B are modified by biotin. The linker is a sequencing linker. In the embodiment of the invention, the joint can be selected according to the specific situation of each sequencing platform, and can be a long joint, a short joint, a bubbling joint or a Y-shaped joint, and the like, which are commonly used in library construction.
In one embodiment, the terminus in the biotinylated linker-terminus complex is selected from a Mosaic terminus.
The biotinylated linker-terminal complex is a double-stranded product formed by annealing a single-linker A-Mosaic terminal forward oligonucleotide shown in SEQ ID No.1 and a reverse oligonucleotide of Mosaic terminal forward oligonucleotide shown in SEQ ID No.3, or a double-stranded product formed by annealing a single-linker B-Mosaic terminal shown in SEQ ID No.2 and a reverse oligonucleotide of Mosaic terminal shown in SEQ ID No. 3.
The difference between the biotinylated transposome formed by the transposase binding to the biotinylated linker-terminus complex and the biotinylated transposome formed by the fusion protein carrying the transposase and the biotinylated linker-terminus complex is that the former does not contain the fusion protein. The two biotinylated transposomes were characterized identically except for the fusion protein, e.g., both transposases were selected from the group consisting of Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn7, Tn8, Tn9, and Tn 10. Preferably, the transposase is selected from Tn 5.
The invention also provides a preparation method of the biotinylated transposome, which comprises the following steps: and (3) incubating the biotinylated linker-terminal complex with the fusion protein carrying the transposase to obtain a biotinylated transposome.
The present invention provides a method for recovering a fragmented nucleic acid product that binds to a target protein, as shown in FIG. 1, comprising the steps of:
1) incubating the nuclei or permeabilized cells with an antibody and/or a biotinylated transposome; directly carrying out enzyme digestion reaction through a biotinylation transposome or a biotinylation transposome guided by an antibody and generating a fragment containing a linker sequence;
2) inactivating the biotinylated transposomes added in step 1) such that biotinylated fragments are released into the solution supernatant;
3) and then adding streptavidin magnetic beads, and recovering to obtain a fragmented nucleic acid product combined with the target protein.
In one embodiment, the recovery method can be used in a CUT & Tag or ATAC-seq experiment. In CUT & Tag, step 1) carrying out enzyme digestion reaction by an antibody and a biotinylated transposome; the biotinylated transposome in ATAC-seq is directly subjected to enzyme digestion reaction.
The cell is a cell that expresses a target protein and a target gene. In one embodiment, the target protein and target gene refer to target proteins and target genes identified in protein-chromatin interaction studies.
In one embodiment, the cell membrane is permeabilized with digitonin. Permeabilized cells facilitate the entry of antibodies into the nucleus where they will bind to histones, transcription factors or cofactors of interest.
In one embodiment, the cells are immobilized with magnetic beads in step 1). The magnetic beads can be combined with glycoprotein on the surface of cell membranes or cell nuclei, and the cells or the cell nuclei can be precipitated by using a magnetic frame.
In one embodiment, the magnetic beads are selected from concanavalin a (cona) magnetic beads. The ConA Magnetic Beads are commercially available, for example NovoNGS Concanavalin A-coated Magnetic Beads.
In one embodiment, the antibody is a ChIP-seq antibody. Such as antibodies targeting histone methylation modifications, RNA polymerase II, and different transcription factors. The antibody includes a primary antibody and a secondary antibody.
In one embodiment, the transposome binds to the antibody in step 1) to form a complex, and the transposase functions to cleave sequences adjacent to the site of DNA that binds to the target protein.
In one embodiment, the biotinylated transposomes are inactivated by addition of SDS in step 2).
In one embodiment, the incubation temperature after adding streptavidin magnetic beads in step 3) is 20-27 ℃.
In one embodiment, the incubation time after adding the streptavidin magnetic beads in step 3) is 20 to 30 minutes.
The invention also provides a construction method of the nucleic acid library, which comprises the step of carrying out PCR amplification on the fragmented nucleic acid products combined with the target protein to obtain the DNA library.
Protein-chromatin interactions can be studied by high throughput sequencing of nucleic acid libraries obtained by the above-described construction methods.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. 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 invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Example one: preparation of biotin adaptor-pAG-Tn5 transposome
1. Incubation of transposase-carrying fusion proteins with biotinylated linkers
The following oligonucleotides were diluted to 200. mu.M in an Annealing buffer (10 mM Tris, 50mM NaCl, 1mM EDTA, pH 8.0), and biotinylated single-stranded A-Mosaic terminal forward oligonucleotide (i.e., ME-A, nucleotide sequence shown in SEQ ID NO. 1) and single-stranded B-Mosaic terminal forward oligonucleotide (i.e., ME-B, nucleotide sequence shown in SEQ ID NO. 2) were annealed to Mosaic terminal Reverse oligonucleotide (ME-Reverse) having nucleotide sequence shown in SEQ ID NO.3 (5' -pCTGTCTTATATACACATCT-NH), respectively2-3') is shown.
The test tube containing ME-A + ME-Reverse and ME-B + ME-Reverse was placed in a 90-95 ℃ heat block for 3-5 minutes, and then the heat block was removed from the heat source and allowed to slowly cool to room temperature (about 45 minutes) to obtain 100. mu.M annealed product. And mixing the double-chain products of ME-A and ME-Reverse formed by 8 mu L100 mu M annealing and the double-chain products of ME-B and ME-Reverse formed by 8 mu L100 mu M annealing with 100 mu L5.5 mu M pAG-Tn 5. The mixture was incubated on a rotary mixer at room temperature for 1 hour and then stored at-20 ℃.
2. Labeling and recovery reaction of biotinylation transposomes
The transposase reaction system is 20 mu L in total, and comprises the following reagents: coli DNA 1. mu.L (total 200 ng), Tn5 transposase [ NovoNGS CUT & Tag 2.0 High-Sensitivity Kit (for Illumina) ] 1. mu.L, 5 XTn 5 reaction solution 4. mu.L, and ultrapure water 14. mu.L.
In Tn5 reaction solution (10 mM Tris,10mM MgCl)2pH 8.0) and a fragmentation reaction using E.coli DNA as a substrate. Fragmentation was performed at 55 ℃ for 5 minutes, 1. mu.L of 2% SDS was added, and heat inactivation was performed at 55 ℃ for 5 minutes. Biotinylated transposomes produced products that were recovered using 1 μ L streptavidin magnetic beads. FIG. 2 is a comparison of the labeling reaction of conventional pAG-Tn5 with biotinylated pAG-Tn5, showing that the transposomes containing the biochemical linkers work equally well with the conventional transposome fragmented genome. FIG. 3 clearly shows that biotinylated fragmentation products are free of nucleic acid bands in the supernatant after incubation with streptavidin magnetic beads. The generated fragmented DNA is completely recovered by streptavidin magnetic beads, namely, the products of the transposon fragmented genome containing the biological linker can be extracted from the system by the streptavidin magnetic beads.
Example two: CUT & Tag Using biotin adaptor-pAG-Tn5 transposome
1. Preparation of cells
The K562 cells were recovered in 10mL RPMI 1640 medium containing 10% FBS, and then placed at 37 ℃ in 5% CO2Concentration in cell culture box. Subculturing every other day at a ratio of 1:2, and stabilizing cell concentration at 0.5 × 106-1.0×106and/mL. To maintain the stability of the experimental cell state.
Centrifuging at 600g at room temperature to collect fresh cells, counting, placing the required number of cells in a new 1.5ml EP tube, centrifuging at 600g at room temperature for 5 minutes, removing supernatant, adding equal volume of Wash Buffer to resuspend and Wash the cells once, and resuspending the cells to ensure that the cell concentration of each reaction is 10 according to the requirement2-105/100μL。
2. Binding of cells to magnetic beads
Vortexing and uniformly mixing the ConA magnetic beads, absorbing the uniformly mixed ConA magnetic beads according to the number of samples, adding 10 times of ConA Binding Buffer in the volume of the original magnetic beads to clean the magnetic beads twice, adding 10 mu L of ConA magnetic beads into each sample, gently blowing, uniformly mixing, placing in a rotary mixer, incubating at room temperature for 10 minutes, and preparing for primary antibody incubation.
3. Incubation of antibodies
The sample tube prepared in the previous step was placed on a magnetic stand and left to stand, the supernatant was removed, and 50. mu.L/sample of a pre-cooled Primary Antibody Buffer [ manufacturer: NovoNGS CUT & Tag 2.0 High-Sensitivity Kit (for Illumina), cat #: n259. Buffers in the following steps are all reagents in the kit, 0.5 mu L of RNAPII Anti { Anti-RNA polymerase II [ CTD 4H8], Ab00832-Absolute Antibody) } is added into each tube of sample, mixed evenly and incubated for 2H at room temperature on a rotary mixer.
After incubation, the cells were allowed to stand on a magnetic frame, the supernatant was removed, a Secondary Antibody (goat anti-mouse IgG Antibody, B103, purchased from Thailand) was diluted 1:200 in a second Antibody Buffer, 100. mu.L of the diluted Secondary Antibody was added to the sample, mixed well and incubated for 1h at room temperature on a rotary mixer. After incubation, the cells were allowed to stand on a magnetic frame, the supernatant was removed and the beads were washed with 500. mu.L of Antibody Buffer and repeated 3 times.
4. Incubation and labeling of turret bodies
The incubated transposable bodies and the standard ChiTag-shaped transposable bodies NovoNGS ChiTag 2.0 Transposome (manufacturer: Shanghai near-shore science and technology Co., Ltd., Cat #: M058) in example I were diluted 1:100 in ChiTag Buffer. The supernatant of the washed substrate in step 3 was removed, 100. mu.L of diluted ChiTag-cassette transposomes were added to each tube of samples, mixed and incubated on a rotary mixer at room temperature for 1 h. After incubation, the cells were allowed to stand on a magnetic frame, the supernatant was removed and the beads were washed with 500. mu.L of Antibody Buffer and repeated 3 times.
Removing the supernatant of the washed substrate, absorbing the supernatant after the magnetic beads are completely separated from the liquid, and taking down the sample tube from the magnetic frame; add 100. mu.L of tagging Buffer to each tube, mix well and incubate for 1h at 37 ℃ on a rotary mixer, this tagged system contains CUT & Tag product.
5. And (3) detection of a reaction result:
adding 2. mu.L of 10% SDS into 100. mu.L of the labeling system, performing heat inactivation at 55 ℃ for 10 minutes, taking 1. mu.L of the labeling system as a substrate to perform PCR detection, wherein the reaction system comprises the following reagents in total of 50. mu.L: a labeling system 1 mu L, Nextra library-establishing primer F/R containing CUT & Tag products (F sequence is shown in SEQ ID NO. 4-SEQ ID NO.7, and R sequence is shown in SEQ ID NO. 8-SEQ ID NO. 11) is 1.5 mu L, 5 x Amplification Mix 10 mu L and ultrapure water 37 mu L respectively.
FIG. 4 shows that the CUT & Tag performed on biotinylated pAG-Tn5 gave similar results to the common transposome, which performed the CUT & Tag on the same biotinated transposome as the common transposome.
Example three: recovery of CUT & Tag products using biotin adaptor-pAG-Tn5 transposome system
Recovery of CUT & Tag products from biotin adaptor-pAG-Tn5 transposome System
And (3) recovering a biotin adaptor-pAG-Tn5 transposome system: for a sample recovered from a biotin adaptor-pAG-Tn5 transposome, 2 muL of 10% SDS is added into each labeling system (100 muL) containing CUT & Tag products, heating is carried out at 55 ℃ for 10 minutes, then 1 muL of washed streptavidin magnetic beads are added into each tube, the mixture is blown and uniformly mixed, the mixture is kept stand at room temperature for 20 minutes, the magnetic beads are combined and then placed on a magnetic frame for 2 minutes, the supernatant is removed, washed by Wash buffer for 2 times, and dried at room temperature. 35 mu.L of TE buffer is redissolved, and the recovered CUT & Tag product is stored at the temperature of 20 ℃ below zero or directly subjected to the next PCR amplification. The recovery process is schematically shown in FIG. 1.
2. Other methods recover the CUT & Tag product
Phenol chloroform extraction-ethanol precipitation: for the sample recovered by ethanol precipitation, adding 3 mu L of 10% SDS, 10 mu L of 0.5M EDTA and 2.5 mu L of 20mg/ml protease K into each CUT & Tag product, performing vortex oscillation, mixing uniformly, and digesting for 1h at 50 ℃ after uniform mixing; add 300. mu.L phenol chloroform mixture to each tube, vortex, mix well, move to phase lock tube, centrifuge at 16,000g for 5 minutes at room temperature. Adding 100 mu L of chloroform into each tube of sample, performing vortex oscillation, mixing well, centrifuging at 16,000g at room temperature for 5 minutes, sucking water phase to a new 1.5ml EP tube, adding 1ml of 100% ethanol, and blowing and mixing well by using a pipette; the sample tubes were left to stand at-20 ℃ for 1h, centrifuged at 4 ℃ for 20 minutes, the centrifuged product was carefully decanted and drained on a paper towel, 1ml of 80% ethanol was added to each tube sample for rinsing, 16,000g was centrifuged at 4 ℃ for 1 minute, the liquid was carefully decanted and drained on a paper towel, and the tube was naturally dried. After drying, 35. mu.L of TE Buffer was added to each tube, and the DNA pellet was resuspended and solubilized by pipetting
And (3) SPRI beads recovery: for samples recovered from SPRI beads and PEG-SPRI beads, 2 μ L of 10% SDS is added into each CUT & Tag product, the product is heated for 10 minutes at 55 ℃, then 200 μ L of magnetic beads after being balanced at room temperature are added into each tube, the mixture is blown and uniformly mixed, the mixture is stood at room temperature for 5 minutes, the magnetic beads are combined and then placed on a magnetic frame to be stood for 5 minutes, the supernatant is removed, the mixture is washed with absolute ethyl alcohol for 2 times, and the mixture is dried at room temperature. Eluted with 35. mu.L of TE buffer, and the recovered CUT & Tag product was stored at-20 ℃ or directly subjected to the next PCR amplification.
And (3) direct PCR treatment: for the direct PCR samples, the supernatant of the fragmentation solution was removed after the CUT & Tag experiment was completed, the samples were washed with 100. mu.L of TAPs Wash buffer, 5. mu.L of 0.2% SDS was added, and the mixture was heated at 70 ℃ for 1 h. The inactivated system was neutralized with SDS by adding 15. mu.L of Triton-X-100. Directly carrying out the next PCR amplification.
PCR library construction and amplification
The PCR reaction procedure was as follows:
1 minute at 72 DEG C
95℃
15 cycles of 95 ℃ for 15s, 60 ℃ for 15s and 72 ℃ for 10s
72℃ 10min
The total volume of the PCR reaction system is 50 mu L, wherein the PCR reaction system comprises 1 mu L, Nextra library-building primers F/R (the F sequence is shown in SEQ ID NO. 4-SEQ ID NO.7, and the R sequence is shown in SEQ ID NO. 8-SEQ ID NO. 11) of the CUT & Tag product, 1.5 mu L, 5 xAmplification Mix 10 mu L and 37 mu L of ultrapure water.
Adding 65 mu L of SPRI beads which are balanced at room temperature into each tube of PCR product, uniformly blowing, standing for 5 minutes at room temperature, placing the combined magnetic beads on a magnetic frame, standing for 5 minutes, removing supernatant, washing for 2 times by using absolute ethyl alcohol, and drying at room temperature. The recovered library was stored at-20 ℃ until second generation sequencing, eluting with 20-40. mu.L of TE buffer.
4.2100 library detection and second generation sequencing
Products were detected by 2100 capillary electrophoresis and the resulting qPCR library molar concentration and 2100 electrophoresis curve were recorded. The sequencing instrument was NovaSeq 6000, and the sequencing amount was approximately 3-10G per sample.
5. Data analysis of results obtained from recovery methods
FIG. 5 is a graph of fragment distribution of the CUT & Tag products obtained by different recovery methods, comparing the fragment distribution obtained by different recovery methods: SA beads (i.e., streptavidin magnetic beads), SPRI beads (i.e., fragment sorting magnetic beads), Ethanol targeting (i.e., phenol chloroform extraction-Ethanol precipitation, or ET for short), Direct PCR (i.e., Direct PCR method, or D-PCR for short), it can be seen that the mainstream SPRI beads extraction causes significant loss of small fragments <150 bp.
FIG. 6 shows the distribution of the CUT & Tag product recovered by SPRI beads and the biotin adaptor-pAG-Tn5 system (i.e., SA beads) on the HPRT1 gene, and the contribution of different size fragments in the CUT & Tag product recovered by SA beads to the results.
FIG. 7 shows the distribution contribution of the CUT & Tag product recovered by the direct PCR method (i.e., D-PCR) and the biotin adaptor-pAG-Tn5 (i.e., SA tags) on the KRAS gene, and the effect of the data amount recovered by different SPRI tags on the result, which shows that the loss of <150bp fragment caused by SPRI tags leads to the loss of signal on the KRAS gene promoter under the condition of low data starting amount in the recovery effect of the SPRI tags and streptavidin magnetic beads on the RNAPII CUT & Tag product.
FIG. 8 is a comparison of the library size generated by four different recovery methods, showing the comparison of recovery efficiency of the four different recovery/treatment methods on the four different CUT & Tag results, with streptavidin magnetic bead (SA beads) recovery being overall a significant advantage.
Example four: recovery of ATAC-seq product by means of biotin adapter-Tn 5-SA Beads system
ATAC-seq experiment
The ATAC-seq process is implemented according to the conventional ATAC-seq processseq experiment. 5X 104 K562 cells were lysed in a pre-cooled lysine buffer (10 mM Tris-HCl, pH7.4, 10mM NaCl, 3mM MgCl2, 0.1% NP40, 0.1% Tween 20, 0.01% Digitonin) in ice for 5min, and nuclei were obtained after low temperature centrifugation. The nuclei were resuspended using a fragmented Mix containing biotinylated Tn5 transposase and reacted on a PCR instrument at 37 ℃ for 30min, and a 50. mu.L reaction system was constructed as follows: ddH2O 17 μ L, 3 XPBS 5 μ L, 10% Tween 200.5 μ L, 0.1% Digitonin 0.5 μ L, 2 XTD buffer 25 μ L, biotinylated Tn5 transposase 2 μ L. Wherein, the 2 XTD Buffer is 20mM Tris-HCl,10mM MgCl2,10% DMF and pH7.4.
After the fragmentation was completed, 2. mu.L of 10% SDS was added to the fragmented product, and the mixture was blown and kneaded, and reacted at 55 ℃ for 5min to terminate the reaction.
ATAC-seq product recovery
And (3) SPRI beads recovery: adding 104 mu L (2 times of the sample volume) of magnetic beads balanced at room temperature into each tube, blowing and uniformly mixing, standing for 5 minutes at room temperature, placing the combined magnetic beads on a magnetic frame for standing for 5 minutes, removing supernatant, washing for 2 times by absolute ethyl alcohol, drying in the air at room temperature (5-10 minutes), adding 35 mu L of sterile water to elute DNA, removing the magnetic beads by using the magnetic frame, and storing the obtained ATAC-seq fragmentation product at-20 ℃ or directly carrying out next-step PCR amplification and library building.
Recovering a biotin adaptor-Tn 5-SA Beads system: adding 1 mu L of washed streptavidin magnetic beads into each tube, blowing and uniformly mixing, standing for 20 minutes at room temperature, placing the combined magnetic beads on a magnetic frame for standing for 2 minutes, removing the supernatant, adding 100 mu L of PBS for rinsing twice, removing the supernatant, adding 35 mu L of sterile water for redissolving the magnetic beads, and storing the recovered ATAC-seq product at-20 ℃ or directly performing the next PCR amplification and library building.
3.2100 library detection and second generation sequencing
2100 capillary electrophoresis detection is carried out on the experimental library, and the molar concentration of the produced qPCR library and a 2100 electrophoresis curve are recorded. The sequencing instrument was NovaSeq 6000, and the sequencing amount was approximately 3-10G per sample.
4. Data analysis of results obtained from recovery methods
FIG. 9 is a graph comparing the fragment distribution of ATAC-seq products obtained from different recovery methods. It can be seen that the mainstream SPRI magnetic bead (i.e. SPRI beads) extraction caused a significant loss of small fragments <150 bp.
FIG. 10 is a graph showing the effect of ATAC-seq products obtained by different recovery methods on a representative region, and it can be seen that there is an additional signal in the recovery of ATAC-seq products from biotin adapt-Tn 5-SA Beads compared with the recovery of SPRI Beads.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the invention set forth herein, as well as variations of the methods of the invention, will be apparent to persons skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
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Claims (10)
1. Use of a biotinylated transposome selected from the group consisting of a biotinylated transposome formed from a fusion protein carrying a transposase and a biotinylated linker-terminus complex capable of binding to the transposase, and/or a biotinylated transposome formed from a transposase bound to a biotinylated linker-terminus complex for recovery of a CUT & Tag or ATAC-seq product; the fusion Protein carrying the transposase also comprises a Protein with the function of binding the Fc segment of the antibody, and the Protein with the function of binding the Fc segment of the antibody is selected from more than two of Protein A, Protein G or Protein L.
2. The use according to claim 1, wherein said transposase is selected from the group consisting of Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn7, Tn8, Tn9, and Tn 10.
3. The use according to claim 1, wherein the fusion protein carrying transposase is pAG-Tn 5.
4. Use according to claim 1, wherein the termini are selected from Mosaic termini.
5. Use according to claim 4, characterized in that the biotinylated linker-terminal complex is a biotinylated linker A-terminal complex and/or a biotinylated linker B-terminal complex.
6. The use according to claim 4, wherein the biotinylated linker-terminal complex is formed after annealing of a single-stranded linker A-Mosaic terminal forward oligonucleotide and a Mosaic terminal reverse oligonucleotide.
7. The use according to claim 4, wherein the biotinylated linker-terminal complex is formed after annealing of a single-stranded linker B-Mosaic terminal forward oligonucleotide and a Mosaic terminal reverse oligonucleotide.
8. A method for recovering a fragmented nucleic acid product that binds to a target protein, the method comprising the steps of:
1) selected from any one of:
1a) incubating the cell nucleus or permeabilized cell with an antibody and a biotinylated transposome; performing an enzymatic cleavage reaction on the biotinylated transposome guided by the antibody and generating a fragment containing the linker sequence;
1b) incubating the nucleus or permeabilized cell with a biotinylated transposome; directly carrying out enzyme digestion reaction through a biotinylated transposome and generating a fragment containing a linker sequence;
2) inactivating the biotinylated transposomes added in step 1) such that biotinylated fragments are released into the solution supernatant;
3) and then adding streptavidin magnetic beads, and recovering to obtain a fragmented nucleic acid product combined with the target protein.
9. A recycling method according to claim 8, characterized in that it further comprises one or several of the following features:
a) the antibody comprises a ChIP-seq antibody;
b) the recovery method can be used in CUT & Tag or ATAC-seq experiments; in CUT & Tag, step 1) carrying out enzyme digestion reaction by an antibody and a biotinylated transposome; carrying out enzyme digestion reaction on a biotinylated transposome in the ATAC-seq directly;
c) the biotinylated transposomes were inactivated by addition of SDS in step 2).
10. A method for constructing a nucleic acid library, comprising subjecting the fragmented nucleic acid product that binds to the target protein and is recovered by the recovery method according to claim 8 or 9 to PCR amplification to obtain a nucleic acid library.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113801915A (en) * | 2021-07-30 | 2021-12-17 | 生物岛实验室 | Method for obtaining target RNA of target RNA binding protein |
| CN114644713A (en) * | 2021-09-03 | 2022-06-21 | 上海爱谱蒂康生物科技有限公司 | Use of biotinylated transposons for identifying and/or enriching chromatin opening region transcription machinery |
| CN115386966A (en) * | 2022-10-26 | 2022-11-25 | 北京寻因生物科技有限公司 | DNA appearance modification library building method, sequencing method and library building kit thereof |
| CN116891532A (en) * | 2022-04-06 | 2023-10-17 | 南京诺唯赞生物科技股份有限公司 | Biotin-avidin-based secondary antibody transposase complex and application thereof in detecting interaction between protein and DNA |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103492588A (en) * | 2011-02-25 | 2014-01-01 | 伊路敏纳公司 | Methods and systems for haplotype determination |
| CN105463066A (en) * | 2014-09-04 | 2016-04-06 | 中国科学院北京基因组研究所 | DNA amplification method |
| CN106507677A (en) * | 2014-04-15 | 2017-03-15 | 伊鲁米那股份有限公司 | For improving insetion sequence bias and increasing the transposase that DNA is input into the modification of tolerance |
| CN107969137A (en) * | 2014-10-17 | 2018-04-27 | 伊卢米纳剑桥有限公司 | Proximity retention swivel base |
| CN108219006A (en) * | 2018-03-27 | 2018-06-29 | 上海欣百诺生物科技有限公司 | A kind of fusion protein of transposase-antibody binding proteins and its preparation and application |
| CN108368540A (en) * | 2015-08-12 | 2018-08-03 | 分子医学研究中心责任有限公司 | The method for studying nucleic acid |
| CN109415758A (en) * | 2017-02-21 | 2019-03-01 | Illumina公司 | The label fragmentation carried out using the swivel base body through immobilization containing connector |
| CN110607352A (en) * | 2019-08-12 | 2019-12-24 | 安诺优达生命科学研究院 | Method for constructing DNA library and application thereof |
| WO2020118543A1 (en) * | 2018-12-12 | 2020-06-18 | 深圳华大生命科学研究院 | Method for separating and/or enriching host source nucleic acid and pathogenic nucleic acid, and reagent and preparation method therefor |
| CN111379031A (en) * | 2018-12-28 | 2020-07-07 | 深圳华大智造科技有限公司 | Nucleic acid library construction method, obtained nucleic acid library and application thereof |
| WO2020144373A1 (en) * | 2019-01-11 | 2020-07-16 | Illumina Cambridge Limited | Complex surface-bound transposome complexes |
| CN111727248A (en) * | 2017-09-25 | 2020-09-29 | 弗雷德哈钦森癌症研究中心 | Efficient targeted in situ whole genome profiling |
-
2021
- 2021-03-23 CN CN202110305115.1A patent/CN112795563A/en active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103492588A (en) * | 2011-02-25 | 2014-01-01 | 伊路敏纳公司 | Methods and systems for haplotype determination |
| CN106507677A (en) * | 2014-04-15 | 2017-03-15 | 伊鲁米那股份有限公司 | For improving insetion sequence bias and increasing the transposase that DNA is input into the modification of tolerance |
| CN105463066A (en) * | 2014-09-04 | 2016-04-06 | 中国科学院北京基因组研究所 | DNA amplification method |
| CN107969137A (en) * | 2014-10-17 | 2018-04-27 | 伊卢米纳剑桥有限公司 | Proximity retention swivel base |
| CN108368540A (en) * | 2015-08-12 | 2018-08-03 | 分子医学研究中心责任有限公司 | The method for studying nucleic acid |
| CN109415758A (en) * | 2017-02-21 | 2019-03-01 | Illumina公司 | The label fragmentation carried out using the swivel base body through immobilization containing connector |
| CN111727248A (en) * | 2017-09-25 | 2020-09-29 | 弗雷德哈钦森癌症研究中心 | Efficient targeted in situ whole genome profiling |
| CN108219006A (en) * | 2018-03-27 | 2018-06-29 | 上海欣百诺生物科技有限公司 | A kind of fusion protein of transposase-antibody binding proteins and its preparation and application |
| WO2020118543A1 (en) * | 2018-12-12 | 2020-06-18 | 深圳华大生命科学研究院 | Method for separating and/or enriching host source nucleic acid and pathogenic nucleic acid, and reagent and preparation method therefor |
| CN111379031A (en) * | 2018-12-28 | 2020-07-07 | 深圳华大智造科技有限公司 | Nucleic acid library construction method, obtained nucleic acid library and application thereof |
| WO2020144373A1 (en) * | 2019-01-11 | 2020-07-16 | Illumina Cambridge Limited | Complex surface-bound transposome complexes |
| CN110607352A (en) * | 2019-08-12 | 2019-12-24 | 安诺优达生命科学研究院 | Method for constructing DNA library and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| FULONG YU ET AL.: "CUT&RUNTools 2.0:A pipeline for single-cell and bulk-level CUT&RUN and CUT&Tag data analysis", 《BIORXIV PREPRINT》 * |
| 韦晔 等: "CUT&Tag产物回收和建库方法的优化", 《遗传》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113801915A (en) * | 2021-07-30 | 2021-12-17 | 生物岛实验室 | Method for obtaining target RNA of target RNA binding protein |
| CN114644713A (en) * | 2021-09-03 | 2022-06-21 | 上海爱谱蒂康生物科技有限公司 | Use of biotinylated transposons for identifying and/or enriching chromatin opening region transcription machinery |
| CN114644713B (en) * | 2021-09-03 | 2023-07-07 | 上海爱谱蒂康生物科技有限公司 | Use of biotinylated transposons for identifying and/or enriching chromatin open region transcription machinery |
| CN116891532A (en) * | 2022-04-06 | 2023-10-17 | 南京诺唯赞生物科技股份有限公司 | Biotin-avidin-based secondary antibody transposase complex and application thereof in detecting interaction between protein and DNA |
| CN115386966A (en) * | 2022-10-26 | 2022-11-25 | 北京寻因生物科技有限公司 | DNA appearance modification library building method, sequencing method and library building kit thereof |
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