Lysis buffer for animal tissue and method for using the same in CUT&Tag

Abstract

The present invention discloses a lysate for animal tissue and a method for using the same in CUT&Tag. The present invention relates to the fields of biotechnology and epigenetic technology, and specifically provides a lysate suitable for animal tissue and a method for using the same in CUT&Tag. The lysate contains an appropriate amount of mild non-ionic detergent NP40 (or CA630) and Triton X-100, which can efficiently lyse tissue clumps while ensuring the integrity of cell nuclei, promote the rapid release of cell nuclei, and thereby obtain cell nuclei suitable for the construction of tissue CUT&Tag libraries. This makes it possible to implement CUT&Tag technology at the animal tissue level, and has great application advantages and high commercial value in the field of epigenetic technology research.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and epigenetic technology, and specifically relates to a lysate for animal tissue and a method for using the same in CUT&Tag. Background Art

[0002] DNA-protein interactions play a crucial role in DNA replication and recombination, RNA transcription, translation, and modification, as well as in gene expression regulation. These interactions are involved in virtually all biological processes. With the introduction of chromatin immunoprecipitation (ChIP-seq) in 2007, it quickly became the gold standard for researchers investigating DNA-protein interactions. However, ChIP-seq requires a large sample and data set, suffers from low signal-to-noise ratio and resolution, and suffers from limitations such as formaldehyde cross-linking, which can alter antigenic epitopes and lead to false-positive results, hindering its further development in epigenomics. CUT&RUN (cleavage under target and release using nuclease), a revolutionary technology in epigenetics, significantly reduces the number of cells required for experiments and enables high signal-to-noise ratio data at low sequencing depth. Therefore, it has become a widely used alternative to ChIP-seq. However, CUT&RUN suffers from high cost, time consumption, and a high workload. In 2019, researchers overcame the limitations of ChIP-seq and CUT&RUN technologies and, based on CUT&RUN, effectively combined ATAC-seq technology to propose CUT&Tag technology. Chromatin targeted cleavage and tagment (Cut&Tag, Cleavage under target and tagment) utilizes Protein A / Protein G and a modified Tn5 transposon to form a fusion protein. Through the interaction between Protein A / Protein G and specific antibodies, Protein A / G-Tn5 is anchored to the specific antibody region to achieve efficient and rapid DNA cleavage. Simultaneously with targeted cleavage, the transposase's ability to catalyze double-stranded recombination and exchange is utilized to tag the DNA with exogenous amplification adapter sequences at both ends, thereby enabling rapid, efficient, and high-resolution sequencing of trace DNA libraries. Compared with ChIP-seq and CUT&RUN technologies, CUT&Tag technology has the advantages of short experimental cycle, simple operation, high signal-to-noise ratio, good repeatability, and low cell input. In the future, it may even be able to achieve single-cell level detection.

[0003] However, despite the mature application of CUT&Tag technology in mammalian cell lines, the extraction of large numbers of intact cell nuclei from animal tissues is still relatively difficult due to the scarcity of tissue samples or the poor preservation of tissues. Therefore, the use of this technology in animal tissue samples is not very widespread. In view of this, the present invention provides a lysis solution formula suitable for extracting cell nuclei from animal tissues and a method for using it in animal tissue CUT&Tag. This invention has important significance and practical value for exploring DNA-protein interaction information in animal tissue samples and for epigenetic research. Summary of the Invention

[0004] In response to the above situation, in order to remedy the above-mentioned existing defects, the present invention provides a lysis solution for animal tissue and a method for using it in CUT&Tag. The lysis solution can effectively solve the existing technical problems. The method has wide applicability, requires a small amount of sample, shortens the experimental time, is easy to operate, and can also be applied to samples in slightly poor preservation conditions.

[0005] The present invention provides the following technical solution: The present invention proposes a lysing solution for animal tissue, which specifically consists of the following components in parts by weight:

[0006]

[0007] The pH of the 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) is 7.5, and the pH of the ethylenediaminetetraacetic acid is 8.0.

[0008] The present invention provides a method for using a lysate for animal tissue in CUT&Tag, which specifically comprises the following steps:

[0009] (1) Weigh the animal tissue sample and resuspend and wash it with DPBS;

[0010] (2) Using a lysis buffer prepared with corresponding weight parts of ethylphenyl polyethylene glycol or octylphenol polyoxyethylene ether, Triton X-100, glycerol solution, 4-hydroxyethylpiperazineethanesulfonic acid, sodium chloride, ethylenediaminetetraacetic acid, protease inhibitors and deionized water, the animal tissue sample was lysed at 4°C, the cell nuclei were initially extracted, a cell nucleus suspension was obtained, and the state of the cell nuclei was observed under a microscope;

[0011] (3) Using a low concentration of 37% formaldehyde to fix the interaction information between DNA and protein in the cell nucleus:

[0012] The cell nuclei were fixed with 37% low-concentration formaldehyde at room temperature for 2 minutes, and cross-linking was terminated with 2.5 M glycine at room temperature for 5 minutes. The nuclei were counted under a microscope, and 200,000 to 300,000 cell nuclei were sampled.

[0013] (4) Preparation of intact cell nuclei: Use one of the following two methods

[0014] ① Incubate the cell nucleus with the treated ConA beads for 15 minutes. The concanavalin-coated ConA beads bind to the polysaccharide of the nuclear membrane to form a complex of ConA beads and cell nucleus. The binding effect of ConA beads and cell nucleus is examined under a microscope. Alternatively, do not use ConA beads and precipitate the cell nucleus by low-speed centrifugation.

[0015] (5) Cell nuclei were incubated with specific antibodies at room temperature for 2 h, or overnight at 4°C for primary antibody treatment;

[0016] (6) The cell nuclei bound to the primary antibody were treated with the corresponding specific antibody for secondary antibody treatment and incubated again at room temperature for 1 hour;

[0017] (7) The cell nuclei bound to the specific antibody are incubated with PA / G-Tn5 transposase; under the precise guidance of the antibody, the transposase targets and cuts the genomic DNA sequence that interacts with the specific antibody and inserts the adapter sequence into both ends of the DNA fragment;

[0018] (8) Add SDS and EDTA to inactivate PA / G-Tn5 transposase; add Proteinase K to digest and extract fragmented DNA;

[0019] (9) Construction of a micro-DNA sequencing library through PCR amplification;

[0020] (10) Screening of sequencing library fragments, library concentration and fragment distribution detection;

[0021] (11) High-throughput sequencing to obtain information on the interaction between DNA and specific proteins.

[0022] Furthermore, the method for fixing the interaction information between DNA and protein in the cell nucleus with low-concentration formaldehyde in step (2) is: after the cell nucleus passes the microscopic examination, the cell nucleus is fixed using single-molecule formaldehyde with an initial concentration of 37% and a final concentration of 0.1%-0.2%.

[0023] Similarly, the lysate provided in this application is also suitable for performing animal tissue ATAC-seq technology.

[0024] The beneficial effects achieved by the present invention using the above structure are as follows: The lysate for animal tissues and the method for using it in CUT&Tag proposed by the present invention have the following advantages and beneficial effects:

[0025] 1. The animal tissue lysis buffer provided by the present invention contains an appropriate amount of mild non-ionic detergent NP40 or CA630, which can effectively lyse tissue clumps, promote the release of cell nuclei, and efficiently obtain cell nuclei suitable for animal tissue CUT&Tag library construction, making it possible to implement CUT&Tag technology at the animal tissue level;

[0026] 2. DNA-protein cross-linking with low-concentration single-molecule formaldehyde fixes the interaction between DNA and protein while avoiding the epitope masking caused by high-concentration formaldehyde cross-linking;

[0027] 3. The present invention has the characteristics of low tissue sample input, simple operation, short experimental cycle, good stability and high reproducibility. From grinding tissue and extracting cell nuclei to incubating antibodies, targeted cleavage and library construction, it can be completed within 2 days;

[0028] 4. The present invention does not require the use of special instruments and can be completed in most laboratories at home and abroad, and can be promoted and used on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0030] Figure 1 Schematic diagram of the animal tissue CUT&Tag principle of the present invention;

[0031] Figure 2 This is the Aglient 4200 quality control image of the DNA library obtained by the Con A beads method of the present invention;

[0032] Figure 3 This is the Aglient 4200 quality control diagram of the DNA library obtained by the centrifugation method of the present invention;

[0033] Figure 4 The size distribution diagram of the reads obtained by sequencing according to the method of the present invention;

[0034] Figure 5 Schematic diagram of the enrichment of peaks obtained by sequencing using the method of the present invention. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0036] It should be noted that the words "front", "rear", "left", "right", "up" and "down" used in the following description refer to directions in the accompanying drawings, and the words "inside" and "outside" refer to directions toward or away from the geometric center of a specific component, respectively.

[0037] The experimental method flow chart of the present invention is shown in Figure 1 .like Figure 1 As shown, after the tissue is ground in liquid nitrogen, lysed with LysisBuffer and fixed with formaldehyde, specific antibodies are targeted and bound to specific proteins, accurately guiding the targeted cutting of PA / G-Tn5 and realizing the construction of animal tissue CUT&Tag library.

[0038] 1. Solution preparation

[0039] 1. Lysis Buffer: Contains 50 mM HEPES (pH 7.5), 140 mM NaCl, 1 mM EDTA (pH 8.0), 10% Glycerol, 0.1%-0.5% NP-40 (or 0.1%-0.5% CA 630), 0.25% Triton X-100, dd H2O, 1× protease inhibitor;

[0040] 2.Binding Buffer: Mix 30ul 10×Binding Buffer and 270ul dd H2O;

[0041] 3. Wash Buffer: 400ul 10× Wash Buffer (-), 80ul 50× protease inhibitor and 3250ul dd H2O, mix well;

[0042] 4. Antibody Buffer: 250 μl Wash Buffer, 1 μl 0.5 M EDTA and 0.8 μl 30% BSA, mix well, prepare immediately before use, and keep on ice;

[0043] 5. 300Buffer: Contains 400ul 10× Dig-300 Buffer(-), 80ul 50× protease inhibitor and 3250ul dd H2O;

[0044] 6. Tagmentation Buffer: Contains 400ul Dig-300 Buffer (-) and 4μl 1M MgCl2. Mix well and prepare before use.

[0045] 2. Experimental Methods and Results

[0046] Experiments were performed using porcine diencephalon tissue.

[0047] 1. Grinding, weighing, and cleaning tissue samples

[0048] (1) Take tissue samples frozen in a -80°C refrigerator;

[0049] (2) Accurately weigh 0.02-0.03 g of tissue sample and place it in a 1.5 ml EP tube;

[0050] (3) Place the EP tube on ice, add 800 μl of pre-chilled DPBS, pipette thoroughly to mix, and resuspend the washed tissue sample. Centrifuge at 2000 g, 4°C, for 5 minutes, discard the supernatant, and retain the pellet.

[0051] 2. Tissue Lysis

[0052] (1) Add 1 ml of Lysis Buffer to the centrifuge tube in step (3) of 1, mix by pipetting on ice for 1 minute, lyse at 4°C for 10 minutes, and mix by pipetting on ice for 1 minute;

[0053] (2) Centrifuge at 2000g for 5 minutes at 4°C, discard the supernatant, retain the precipitate, and immediately resuspend the precipitate in 1 ml of pre-chilled DPBS to obtain a cell nucleus suspension.

[0054] 3. Microscopic examination

[0055] The tissue lysis effect and the status of the cell nucleus were observed under a microscope.

[0056] 4. Formaldehyde fixation and cross-linking

[0057] (1) Add 37% monomolecular formaldehyde (final concentration 0.1-0.2%) to the 2(2) cell nucleus suspension and fix at room temperature for 2 minutes;

[0058] (2) Add 2.5 M glycine to terminate cross-linking (final concentration 0.125 M);

[0059] (3) Centrifuge at 2000 g, 4°C for 5 min, discard the supernatant, and retain the precipitate to obtain the cell nuclei fixed with low concentration formaldehyde.

[0060] 5. Wash cell nuclei, count and take cells

[0061] (1) Add 1 ml of pre-cooled DPBS to the precipitate from step 4(3) and mix thoroughly by pipetting; centrifuge at 2000 g, 4°C for 5 min, discard the supernatant, and keep the precipitate;

[0062] (2) Resuspend the precipitate obtained in step 5(1) in 1 ml of DPBS, mix thoroughly, take 9 μl of the cell suspension, add 1 μl of trypan blue dye, and count the cells using a cell counter;

[0063] (3) After counting, take 300,000 cells from 5(1), transfer them to a 1.5 ml Low Binding EP tube, centrifuge at 2000 g, 4°C for 5 min, discard the supernatant, and keep the pellet;

[0064] 6. Resuspend the cell nuclei in Wash buffer

[0065] Add 500 μl of Wash buffer to the EP tube in step (3) of step 5 and mix thoroughly; centrifuge at 2000 g, 4°C for 5 min, discard the supernatant, and retain the precipitate;

[0066] Next, choose one of 7-13 or 14-18 to perform the operation

[0067] 7. Resuspend the cell nuclei in Wash buffer

[0068] Resuspend the pellet obtained in 6% slurry with 100ul Binding buffer.

[0069] 8. Prepare Con A beads

[0070] (1) Transfer 10 μl of Con A beads / sample to a 1.5 ml Low Binding EP tube, place on a magnetic separator, and remove the supernatant.

[0071] (2) Add 100-200 μl of Binding buffer to step 8(1), wash the Con A beads, place on a magnetic plate, and remove the supernatant. Repeat this step 2-3 times.

[0072] (3) Add 10 μl of binding buffer per sample and resuspend the Con A beads.

[0073] 9. Incubate cell nuclei and Con A beads

[0074] (1) Resuspend the cell nuclei in Wash buffer 7 and place them on a thermomixer (25°C, 1100 rpm, 15s on, 2min off). Add the treated Con A beads while shaking and incubate for 20-30min.

[0075] (2) Take 1 μl of cell nuclei and incubate the suspension with Con A beads, and examine the binding efficiency between the cell nuclei and Con A beads under a microscope;

[0076] (3) Place the EP tube on a magnetic rack, wait for the liquid to clarify, and discard the supernatant.

[0077] 10. Primary Antibody Incubation

[0078] (1) Dilute the primary antibody according to 100ul / sample Antibody buffe. The antibody dosage should refer to the immunoassay concentration recommended in the antibody instructions (it is generally recommended to dilute the primary antibody at 1:100).

[0079] (2) Add the mixed antibody to the conjugate of Con A beads and cell nuclei obtained in step 9(3);

[0080] (3) Incubate the plate on a thermomixer (25°C, 900 rpm, 15 seconds on, 2 minutes off) for 2-3 hours at room temperature, or overnight at 4°C.

[0081] 11. Secondary Antibody Incubation

[0082] (1) Dilute the secondary antibody to 100 μl / sample in Wash Buffer and mix thoroughly (it is generally recommended to dilute the secondary antibody at 1:100).

[0083] (2) Place the EP tube on a magnetic field and discard the supernatant after the liquid becomes clear.

[0084] (3) Add the diluted secondary antibody to the EP tube and gently tap to mix;

[0085] (4) Incubate at room temperature for 1 hour on a thermomixer (25°C, 900 rpm, 15 seconds on, 2 minutes off);

[0086] (5) Centrifuge briefly, place the EP tube on a magnetic field, wait for the liquid to become clear, and discard the supernatant.

[0087] (6) Add 500ul–600ul of Wash Buffer to the EP tube and flick gently to mix. Centrifuge briefly and place the EP tube on a magnetic rack. Once the liquid has clarified, discard the supernatant. Repeat this washing step three times.

[0088] (7) After the final wash, do not remove the Wash Buffer to prevent the Con A beads from drying out too much.

[0089] 12.PA / G-Tn 5 transposase incubation

[0090] (1) Dilute PA / G-Tn 5 (0.04 μM per reaction) in 300 μL Buffer at 100 μL / sample and vortex to mix.

[0091] (2) Discard the Wash Buffer in the 12(7)EP tube;

[0092] (3) Add 100 μl of diluted PA / G-Tn 5 per sample and gently mix the PA / G-Tn 5 and Con A beads. Incubate at room temperature with shaking for 1 hour.

[0093] (4) Centrifuge briefly, place the EP tube on a magnetic stand for 2 minutes, and discard the supernatant;

[0094] (5) Add 500–600 μl of 300 Buffer to the EP tube and flick gently to mix. Centrifuge briefly and place the EP tube on a magnetic plate. Once the liquid has clarified, discard the supernatant. Repeat this washing procedure three times.

[0095] 13.PA / G-Tn 5 transposase activation, cleavage

[0096] (1) Centrifuge briefly, place the EP tube on a magnetic rack for 2 minutes, and discard the supernatant.

[0097] (2) Add 300 μl of Tagmentation Buffer to the EP tube and gently tap to mix.

[0098] (3) Incubate at 37°C with shaking for 1 hour.

[0099] 14. Incubation with primary antibody

[0100] (1) Dilute the primary antibody according to 100ul / sample Antibody buffe. The antibody dosage should refer to the immunoassay concentration recommended in the antibody instructions (it is generally recommended to dilute the primary antibody at 1:100).

[0101] (2) Mix the mixed antibodies with the cell nuclei in step 6;

[0102] (3) Incubate with rotation at room temperature for 2-3 hours, or at 4°C overnight.

[0103] 15. Incubation with secondary antibody

[0104] (1) Dilute the secondary antibody to 100 μl / sample in Wash Buffer and mix thoroughly (it is generally recommended to dilute the secondary antibody at 1:100).

[0105] (2) Centrifuge the cell nucleus suspension incubated with the primary antibody in step 15(3) at 2000 g at 4°C for 5-10 min, discard the supernatant, and retain the precipitate;

[0106] (3) Add the diluted secondary antibody to the EP tube and gently tap to mix;

[0107] (4) Incubate at room temperature for 1 hour;

[0108] (5) Centrifuge at 2000 g for 5-10 min at 4°C and remove the supernatant.

[0109] (6) Add 500–600 μl of Wash Buffer to the EP tube and gently flick to mix. Centrifuge at 2000 g, 4°C, for 5–10 min. Discard the supernatant and retain the pellet. Repeat this washing procedure three times.

[0110] 16.PA / G-Tn 5 transposase incubation

[0111] (1) Dilute PG / A-Tn 5 (0.04 μM per reaction) in 300 μL Buffer at 100 μL / sample and vortex to mix.

[0112] (2) Add 100 μl of diluted PG / A-Tn 5 to each sample and gently mix the PG / A-Tn 5 and Con A beads. Incubate at room temperature with shaking for 1 hour.

[0113] (3) Centrifuge at 2000 g, 4°C for 5-10 min and remove the supernatant.

[0114] (4) Add 500–600 μl of 300 Buffer to the EP tube and gently flick to mix. Centrifuge at 2000 g, 4°C, for 5–10 min. Discard the supernatant and retain the precipitate. Repeat this washing procedure three times.

[0115] 17.PG / A-Tn 5 transposase activation, cutting

[0116] (1) Centrifuge at 2000 g, 4°C for 5-10 min and remove the supernatant.

[0117] (2) Add 400 μL of Tagmentation Buffer to the EP tube and gently tap to mix.

[0118] (3) Incubate at 37°C with shaking for 1 hour;

[0119] (4) Add 13.5ul 0.5M EDTA, 4ul 10% SDS and 20-30ul 800U / ml Proteinase K to each sample to terminate the cleavage.

[0120] 18.DNA Extraction

[0121] (1) Digest the sample from step (4) in 17 at 50°C / 55°C overnight with shaking;

[0122] (2) Prepare MAXTRACT separation gel and centrifuge at 15,000 rpm for 1 min.

[0123] (3) Add 400 μl / sample of phenol:chloroform:isoamyl alcohol (25:24:1) and shake vigorously until the solution turns milky white;

[0124] (4) Centrifugation at 15,000 rpm for 5 min at 4°C;

[0125] (5) Transfer the supernatant to a new 1.5 ml centrifuge tube. Add 16 μl of 5 M NaCl and 2 μl of glycogen and mix thoroughly.

[0126] (6) Add 920 μl of 100% icy ethanol, mix well, and precipitate at -80°C for 2 hours;

[0127] (7) Centrifuge at 15,000 rpm at 4°C for 30-40 min, discard the supernatant, and retain the precipitate;

[0128] (8) Add 1 ml of 75% cold ethanol to the precipitate, mix well, and wash the precipitate;

[0129] (9) Centrifuge at 4°C, 15,000 rpm for 5-10 min, discard the supernatant, and retain the precipitate. Allow the ethanol to evaporate completely and allow the mixture to dry completely.

[0130] (10) Add 20-30 μl of RNAase-free water to the sample to fully dissolve the precipitate;

[0131] (11) After the concentration of the purified DNA sample is determined by Qbit, the sample can be directly used for PCR library amplification or stored at -20°C.

[0132] 19. Library Construction and Amplification

[0133] (1) Take 200ul of RNase-free EP tube and prepare the reaction system. The reaction system is as follows:

[0134]

[0135] Repeatedly pipette the above reaction system 20 times to mix thoroughly, and centrifuge instantly. This step is important!

[0136] Primer was synthesized based on the Illumina sequencing platform-specific Primer.

[0137] (2) PCR amplification conditions are as follows:

[0138]

[0139] 20. Screen library fragments and perform library quality control as needed

[0140] (1) 0.55X KAPA Pure beads screened out fragments below 800 bp, and 0.35X KAPA Pure beads screened out fragments above 200 bp;

[0141] (2) Qbit was used to determine the library concentration, and Agilent 2100 Bioanalyzer was used to detect the library length distribution;

[0142] (3) Based on the quality control of the library, 1.3X KAPA Pure beads were used to remove excess primers and the concentration was determined by Qbit.

[0143] 21. Computer test

[0144] like Figure 2 As shown, the peak diagram of Aglient 4200 for animal tissue CUT&Tag using Con A beads shows that the fragment distribution is reasonable.

[0145] like Figure 3 As shown, without using Con A beads, the Aglient 4200 peak diagram of animal tissue CUT&Tag was collected by centrifugation, showing that the fragment distribution is reasonable.

[0146] like Figure 4 As shown, the size distribution of reads obtained by sequencing using the method of the present invention is reasonable.

[0147] like Figure 5 As shown, peaks have similar enrichment among different samples sequenced by the method of the present invention.

[0148] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, material, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, material, or apparatus.

[0149] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A use of a lysate in extracting animal tissue cell nuclei, characterized in that: The lysate specifically consists of the following components in parts by weight: The pH of the 4-hydroxyethylpiperazineethanesulfonic acid is 7.5, and the pH of the ethylenediaminetetraacetic acid is 8.

0.

2. Use of the lysate in the application according to claim 1 in CUT&Tag, characterized in that: The specific steps include: (1) Weigh the animal tissue sample and resuspend and wash it with DPBS; (2) Using a lysis buffer prepared with corresponding weight proportions of ethylphenyl polyethylene glycol or octylphenyl-polyethylene glycol, Triton X-100, glycerol solution, 4-hydroxyethylpiperazineethanesulfonic acid, sodium chloride, ethylenediaminetetraacetic acid, protease inhibitors and deionized water, the animal tissue sample was lysed at 4°C, the cell nuclei were initially extracted, a cell nucleus suspension was obtained, and the state of the cell nuclei was observed under a microscope; (3) Fix the DNA-protein interaction information in the cell nucleus with monomolecular formaldehyde at an initial concentration of 37% and a final concentration of 0.1%-0.2%: Fix the cell nucleus with monomolecular formaldehyde at an initial concentration of 37% and a final concentration of 0.1%-0.2% at room temperature for 2 minutes, terminate the cross-linking with 2.5M glycine at room temperature for 5 minutes, count the nuclei under a microscope, and collect 200,000-300,000 cell nuclei; (4) Preparation of intact cell nuclei: Use one of the following two methods ① Incubate the cell nucleus with the treated ConA beads for 15 minutes. The concanavalin-coated ConA beads bind to the polysaccharide of the nuclear membrane to form a complex of Con A beads and the cell nucleus. The binding effect of Con A beads and the cell nucleus is examined under a microscope. Alternatively, do not use Con A beads and precipitate the cell nucleus by low-speed centrifugation. (5) Cell nuclei were incubated with specific antibodies at room temperature for 2 h, or overnight at 4°C for primary antibody treatment; (6) The cell nuclei bound to the primary antibody were treated with the corresponding specific antibody for secondary antibody treatment and incubated again at room temperature for 1 hour; (7) The cell nuclei bound to the specific antibody are incubated with PA / G-Tn5 transposase; under the precise guidance of the antibody, the transposase targets and cuts the genomic DNA sequence that interacts with the specific antibody and inserts the adapter sequence into both ends of the DNA fragment; (8) Add SDS and EDTA to inactivate PA / G-Tn5 transposase; add Proteinase K to digest and extract fragmented DNA; (9) Construction of a micro-DNA sequencing library through PCR amplification; (10) Screening of sequencing library fragments, library concentration and fragment distribution detection; (11) High-throughput sequencing to obtain information on the interaction between DNA and specific proteins.

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