A high-throughput tissue sample chromatin immunoprecipitation combined with chromatin conformation capture method

Abstract

The present invention discloses a high-throughput tissue sample chromatin immunoprecipitation combined with chromatin conformation capture method, which can effectively enrich the DNA after chromatin conformation capture combined with chromatin immunoprecipitation steps in complex clinical tissue samples, and the extracted DNA is sufficient for subsequent library construction. The present invention independently proposes a series of processing procedures such as liquid nitrogen quick freezing and grinding pretreatment, combined enzymatic digestion, and physical fragmentation during cross-linking. Small molecule drugs that maintain cell viability are added during the process, so that cells can be fully dissociated and interstitial tissue can be removed, while retaining living cells in the tissue for subsequent steps to the maximum extent; the first tissue processing technology for "chromatin immunoprecipitation combined with chromatin conformation capture experiment" was created. The present invention is not only suitable for complex tissue samples, but also suitable for drawing specific protein-mediated chromatin three-dimensional interaction maps in cell lines cultured in vitro after a slight modification, and the efficiency is greatly improved compared with existing similar technologies.

Description

Technical Field

[0001] The present invention relates to the detection of higher-order chromatin structures in complex biological tissues, and more specifically, to a method for high-throughput detection of three-dimensional chromatin interactions mediated by specific proteins in tissue samples by combining chromatin immunoprecipitation with chromatin conformation capture technology. Background Art

[0002] Chromatin sites that are not linearly adjacent may approach and bind to each other in the three-dimensional space of the cell nucleus. This phenomenon is called three-dimensional chromatin interaction. The three-dimensional interaction of chromatin plays an important regulatory role in gene expression by remotely binding the genomic enhancer region with the gene promoter region, and plays an important role in the development of individuals and the occurrence of diseases. Many different types of proteins are involved in mediating the formation of three-dimensional chromatin interaction structures, such as CTCF and MED1. In situ Hi-C technology can capture all chromatin interactions in cells with high throughput, which is of great value for understanding the higher-level structure of chromatin, but this technology cannot distinguish the three-dimensional chromatin interactions mediated by a specific protein (Lieberman-Aiden, et al., 2009, Science, PMID: 19815776). HiChIP technology (Mumbach, et al., 2016, Nature Methods, PMID: 27643841) and ChIA-PET technology (Fullwood, et al., 2009, Nature, PMID: 19890323) combine specific protein-mediated chromatin immunoprecipitation technology with chromatin conformation capture technology to specifically identify three-dimensional chromatin interactions mediated by a certain protein.

[0003] However, since chromatin immunoprecipitation technology and chromatin conformation capture technology are inherently complex and involve many steps, many intermediate steps will gradually reduce the yield of the final target DNA, resulting in the high requirements for the starting cell amount for HiChIP technology and ChIA-PET technology. They can only be performed in cell lines cultured in vitro and cannot be used to capture the three-dimensional chromatin interactions mediated by specific proteins in clinical tissue samples. Describing the long-range chromatin interactions mediated by specific proteins during the development of the disease is of great significance for understanding the pathogenic mechanism and discovering therapeutic targets, but no technology has been reported to achieve this goal in clinical tissue samples (such as tumors and paracancerous tissues). Since clinical samples are composed of complex cell types, including connective tissue and various types of cells, almost all steps in HiChIP or ChIA-PET technology, such as restriction endonuclease digestion conditions, ultrasonic conditions, immunoprecipitation process, library amplification methods, etc., are not applicable and need to be re-explored and optimized. In addition, the method of tissue fragmentation at the beginning of the experiment is crucial to the success or failure of subsequent experiments, and the best method needs to be determined. Summary of the invention

[0004] In view of the defect that the existing high-throughput technology for mapping three-dimensional chromatin interactions mediated by specific proteins is not suitable for complex clinical tissue samples, the present invention proposes a high-throughput tissue sample chromatin immunoprecipitation combined with chromatin conformation capture method.

[0005] The present invention is achieved through the following technical solution: a high-throughput tissue sample chromatin immunoprecipitation combined with chromatin conformation capture method, comprising the following steps:

[0006] (1) Liquid nitrogen quick freezing, grinding and enzymatic hydrolysis of tissue

[0007] The tissue was snap-frozen using liquid nitrogen, followed by enzymatic digestion using an enzymatic digestion solution, followed by TrypLE digestion; the enzymatically digested cells were then cross-linked using 10 mL of 1% paraformaldehyde, and cross-linking was terminated using a Glycine solution;

[0008] (2) Tissue lysis

[0009] Add chromatin conformation capture lysis buffer, which contains 50 mM Hepes-KOH pH7.5, 140 mM NaCl, 1 mM EDTA, 0.5% Igepal CA-630; 0.25% Triton X-100, and protease inhibitors, and rotate and lyse for 50 minutes;

[0010] (3) Enzyme digestion

[0011] Tissue lysates were digested with restriction endonuclease MboI;

[0012] (4) Biotin labeling and ligation

[0013] Add biotin-dATP, dCTP, dGTP, dTTP, and DNA polymerase to the digested product, label at 37°C for 1 hour; then add ligation solution and ligate at room temperature for 4 hours;

[0014] (5) Ultrasound

[0015] One day before sonication, the antibody is pre-bound to the immunomagnetic beads, and the ligation product obtained in step (4) is resuspended in sonication buffer and sonicated in a sonicator;

[0016] (6) Chromatin immunoprecipitation

[0017] The antibody-magnetic beads pretreated in step (5) were mixed with the ultrasonic product and rotated overnight at 4°C; after overnight, the magnetic beads were washed with a washing buffer solution and an ammonium bicarbonate solution respectively;

[0018] (7) DNA extraction

[0019] Elute the DNA on the magnetic beads using a DNA elution buffer, and then extract the DNA using a DNA purification kit;

[0020] (8) Biotin affinity and transposase cleavage

[0021] Resuspend the Streptavidin-C magnetic beads in reaction buffer, and add the DNA solution obtained in step (7) to the Streptavidin-C magnetic bead suspension, and perform affinity at room temperature for 30 minutes; add reaction buffer and Tn5 transposase to the Streptavidin-C magnetic beads bound to the biotin-labeled DNA, and cut at 55°C for 10 minutes with interval shaking;

[0022] (9) Library construction

[0023] First, use Phusion high-fidelity DNA polymerase reaction solution to pre-clean the Streptavidin-C magnetic beads obtained in step (8). Use PCR amplification solution to amplify the DNA on the Streptavidin-C magnetic beads. Set the PCR instrument to preheat at 72°C in advance to ensure that the reaction solution heats up quickly. The PCR amplification program is: 72°C 5 minutes -> 98°C 1 minute -> 98°C 15 seconds -> 63°C 30 seconds -> 72°C 1 minute, 9 to 12 cycles. Set the PCR instrument to preheat at 72°C before officially starting the PCR amplification program (because the quality of tissue-derived DNA is not as good as that of cell line-derived DNA, if the PCR program is started at room temperature without preheating, there is a high probability that the amplification will not be successful in the first time, and the quality of the library after the second amplification is very different from that of the library that succeeded in the first amplification). Perform library electrophoresis on the Agilent TapeStation to identify the quality of library amplification; use the Pippin DNA Size Selection system or Ampure XP magnetic beads to screen 200-700bp library fragments.

[0024] Furthermore, in the step (1), the enzymatic solution is 100 U / ml type II collagenase, 20 nM ROCK inhibitor Y-27632, and 1× antibacterial-antifungal agent, and the above components are dissolved in 1× Hank's balanced salt solution; the enzymatic hydrolysis conditions are rotating enzymatic hydrolysis at 37°C for 2 hours; polyformaldehyde cross-linking the enzymatically hydrolyzed cells for 10 minutes, and the tissue homogenizer is used to grind 3 times at level 1, each time for 6 seconds.

[0025] Furthermore, in step (3), the restriction endonuclease MboI digestion conditions are 37° C. for 2 hours.

[0026] Furthermore, in step (4), the ligation solution contains 300 μL 10X NEB T4 DNA ligase buffer, 250 μL 10% Triton X-100, 6 μL 50 mg / mL BSA, 20 μL 400 U / μL T4 DNA ligase, and 1320 μL triply distilled water.

[0027] Furthermore, in step (5), the antibody and immunomagnetic beads are Protein-A and Protein-G magnetic beads respectively, and the ultrasonic buffer is 10 mM Tris-HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Na-Deoxycholate; 0.5% N-lauroylsarcosine; protease inhibitors are added immediately after use; the ultrasonic condition is 10 seconds on + 30 seconds off for each cycle, and 10 cycles are repeated. The ultrasonic tube is taken out and shaken to mix, and then put back, and 10 cycles are continued.

[0028] Furthermore, in step (6), the washing buffer is 50 mM Tris, pH 7.6; 150 mM NaCl; 1 mM EDTA; 0.1% SDS; 1% Igepal CA630 (or NP40); 0.5% Na-Deoxycholate; and protease inhibitors are added immediately after use.

[0029] Furthermore, in step (7), the elution step is: rotate at room temperature for 10 minutes, shake on a hot plate at 37°C for 3 minutes, transfer the supernatant to a new tube, add proteinase K, shake on a hot plate at 55°C for 45 minutes; shake on a hot plate at 67°C for 3.5 hours.

[0030] Furthermore, in step (8), the reaction buffer is 20 mM Tris-HCl pH 7.5; 10 mM MgCl2; 20% Dimethylformamide.

[0031] Furthermore, in step (9), the PCR amplification solution contains 25 μL 2X Phusion high-fidelity DNA polymerase reaction solution, 1 μL 12.5 μM universal amplification primer, 1 μL 12.5 μM barcode-specific amplification primer, and 23 μL triply distilled water; the PCR amplification program is: 72°C 5 minutes -> 98°C 1 minute -> 98°C 15 seconds -> 63°C 30 seconds -> 72°C 1 minute, 9 to 12 cycles.

[0032] The present invention replaces steps (1) and (2) with a simplified cell lysis process, and the subsequent steps are completely consistent. It can be applied to draw a three-dimensional chromatin interaction map mediated by a specific protein in a cell line cultured in vitro.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The present invention can effectively enrich DNA after chromatin conformation capture and chromatin immunoprecipitation steps in complex clinical tissue samples. That is, after DNA extraction in step (7), generally 200-500 ng of DNA can be obtained from 20-50 mg of tissue, which is sufficient for subsequent library construction. However, the existing technology cannot obtain DNA from tissues through similar steps.

[0035] (2) The present invention independently proposed a series of processing procedures, including liquid nitrogen quick-freezing and grinding pretreatment, combined enzymatic digestion, and physical fragmentation during the cross-linking process. During the process, the small molecule drug ROCK inhibitor Y-27632 was added to maintain cell viability, thereby fully dissociating cells and removing interstitial tissues, while retaining living cells in the tissue to the maximum extent for subsequent steps. The present invention is the first tissue processing technology used for "chromatin immunoprecipitation combined with chromatin conformation capture experiment". Currently, no other tissue processing technology has been found in the literature to be applied to "chromatin immunoprecipitation combined with chromatin conformation capture experiment".

[0036] (3) In steps (3)-(4) of the present invention, the chromatin conformation capture lysis buffer proposed by us is used to extract the cell nucleus, and the enzyme cutting and connection conditions are optimized, so that the spatially adjacent DNA in the chromatin complex can be fully cut and randomly connected; the ultrasonic conditions in step (5) of the present invention and the immunoprecipitation method in step (6) are both step innovations made at the experimental principle level. Compared with the existing steps using the ultrasonic / immunoprecipitation buffer containing Triton X-100, we use the ultrasonic / immunoprecipitation buffer containing sodium deoxycholate and N-lauroylsarcosine, which can fully permeate the cell nuclear membrane and fully dissolve the protein-chromatin complex in the cell nucleus, and at the same time provide suitable buffer conditions for antigen-antibody binding, which is one of the keys to improving the DNA yield of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention will be further described below in conjunction with the accompanying drawings.

[0038] Figure 1 The electrophoresis diagram of the TapeStation 4200 library of the present invention;

[0039] Figure 2 This is a quality control chart of the original sequencing data by the hicup software of the present invention;

[0040] Figure 3This is an APA analysis diagram of chromatin loops mediated by CTCF of the present invention;

[0041] Figure 4 This is the three-dimensional chromatin interaction map (chr8) mediated by CTCF of the present invention. DETAILED DESCRIPTION

[0042] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative, rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, without departing from the essence and scope of the present invention, various changes or modifications to the material composition and dosage in these embodiments also belong to the protection scope of the present invention. The present invention is further described below in conjunction with specific examples.

[0043] Example 1

[0044] The various steps included in the present invention and the core scheme of each step are as follows:

[0045] (1) Liquid nitrogen quick-frozen tissue grinding and enzymatic hydrolysis: First, the tissue was quick-frozen with liquid nitrogen, and then the tissue was hydrolyzed with an enzymatic solution (100 U / ml type II collagenase; 20 nM ROCK inhibitor Y-27632; 1× antibacterial-antifungal agent; the above components were dissolved in 1× Hank's balanced salt solution) at 37°C for 2 hours. Then, 0.25% TrypLE was used for hydrolysis for 5 minutes. The hydrolyzed cells were cross-linked with 10 mL of 1% paraformaldehyde for 10 minutes. During the process, a tissue homogenizer (Bio-Gen PRO200) was used for grinding 3 times at level 1, each time for 6 seconds. The cross-linking was terminated with a glycocine solution.

[0046] (2) Tissue lysis: First add 1 mL of chromatin conformation capture lysis buffer (containing 50 mM Hepes–KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5% Igepal CA-630; 0.25% Triton X-100, 10% glycerol, and protease inhibitors) and rotate for 50 minutes.

[0047] (3) Enzyme digestion: The tissue lysate was digested with 1125 U of restriction endonuclease MboI (NEB-R0147) at 37°C for 2 h.

[0048] (4) Biotin labeling and ligation: Add 75 μL 0.4 mM biotin-dATP (Thermo19524016), 3 μL 10 mM dCTP, 3 μL 10 mM dGTP, 3 μL 10 mM dTTP, and 20 μL 5 U / μL DNA polymerase I (NEB-M0210) to the digested product and label at 37°C for 1 hour; add 1896 μL of the prepared ligation solution (containing 300 μL 10X NEB T4 DNA ligase buffer (NEB-B0202), 250 μL 10% Triton X-100, 6 μL 50 mg / mL BSA, 20 μL 400 U / μL T4 DNA ligase (NEB-M0202), and 1320 μL triply distilled water) and ligate at room temperature for 4 hours.

[0049] (5) Ultrasound: One day before ultrasound, the antibody was pre-bound to the immunomagnetic beads, that is, 40 μL of Protein-A was mixed with 40 μL of Protein-G magnetic beads (Thermofisher) and rotated with 7.5 μg of the corresponding antibody at 4°C overnight. The ligation product obtained in step (4) was resuspended in ultrasound buffer (10 mM Tris–HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Na–Deoxycholate; 0.5% N-lauroylsarcosine; protease inhibitors were added immediately before use) and ultrasound was performed in a Bioruptor® Pico ultrasound machine, with each cycle of 10 seconds on + 30 seconds off. Repeat 10 cycles, remove the ultrasound tube, shake to mix, and put it back, and continue 10 cycles.

[0050] (6) Chromatin immunoprecipitation: Mix the antibody-magnetic beads pretreated in step (5) with the ultrasonic product and rotate overnight at 4°C; after overnight, wash the magnetic beads 10 times with washing buffer (50 mM Tris, pH 7.6; 150 mM NaCl; 1 mM EDTA; 0.1% SDS; 1% Igepal CA630 (or NP40); 0.5% Na-Deoxycholate; protease inhibitors added immediately before use); wash the magnetic beads once with 100 mM ammonium bicarbonate solution.

[0051] It is noteworthy that the ultrasonic conditions in step (5) and the immunoprecipitation method in step (6) of the present invention are both step innovations made from the experimental principle level. Compared with the existing steps using an ultrasonic / immunoprecipitation buffer containing Triton X-100, we use an ultrasonic / immunoprecipitation buffer containing sodium deoxycholate and N-lauroylsarcosine, which can fully permeate the nuclear membrane and fully dissolve the protein-chromatin complex in the nucleus, while providing suitable buffer conditions for antigen-antibody binding, which is one of the keys to improving the DNA yield of the present invention.

[0052] (7) DNA extraction: Use DNA elution buffer (freshly prepared: 0.1% SDS; 50 mM NaHCO3) to elute the DNA on the magnetic beads (elution steps: rotate at room temperature for 10 minutes, shake on a hot plate at 37°C for 3 minutes, transfer the supernatant to a new tube, add proteinase K, shake on a hot plate at 55°C for 45 minutes; shake on a hot plate at 67°C for 3.5 hours), and use a DNA purification kit (Zymo) to extract the DNA.

[0053] (8) Biotin affinity and transposase cleavage: Resuspend the Streptavidin-C magnetic beads in reaction buffer (20 mM Tris-HCl pH 7.5; 10 mM MgCl2; 20% Dimethylformamide), and add the DNA solution obtained in step (7) to the Streptavidin-C magnetic bead suspension for affinity at room temperature for 30 minutes; add reaction buffer and an appropriate amount of Tn5 transposase (for example, add 0.8 μL Tn5 if the amount of DNA extracted in step (7) is 25 ng) to the Streptavidin-C magnetic beads bound to the biotin-labeled DNA, and cleave at 55°C for 10 minutes with interval shaking. The amount of Tn5 transposase used was determined through trial and error, and the ratio of 0.8 μL Tn5 per 25 ng DNA can make the DNA fragments after cleavage concentrated in the range of 200-700 bp.

[0054] (9) Library construction: First, pre-clean the Streptavidin-C magnetic beads obtained in step (8) with Phusion high-fidelity DNA polymerase reaction solution (NEB m0531s), and use PCR amplification solution (containing 25 μL 2X Phusion high-fidelity DNA polymerase reaction solution, 1 μL 12.5 μM universal amplification primer, 1 μL 12.5 μM barcode-specific amplification primer, and 23 μL triply distilled water) to amplify the DNA on the Streptavidin-C magnetic beads. Preheat the PCR instrument to 72°C in advance, and then perform the PCR amplification program: 72°C 5 minutes -> 98°C 1 minute -> (98°C 15 seconds -> 63°C 30 seconds -> 72°C 1 minute) for 9 to 12 cycles; perform library electrophoresis on the Agilent TapeStation to identify the quality of library amplification; use the PippinDNA Size Selection System or Ampure XP magnetic beads to screen 200-700 bp library fragments.

[0055] In this step, the PCR amplification pre-treatment is optimized to ensure that the final DNA template derived from the tissue sample can achieve the best effect after the first amplification. If the tissue-derived DNA is directly PCR amplified without the pre-treatment step of the present invention, it is likely that the amplification will not be successful, and repeated amplification will lead to a serious decrease in library quality, that is, an increase in the ratio of PCR duplicate fragments.

[0056] Example 2

[0057] The present invention is not only applicable to complex tissue samples, but also, after a slight modification (the tissue fragmentation and lysis process can be replaced with a simplified cell lysis process, and the subsequent steps are completely the same), is also applicable to mapping specific protein-mediated chromatin three-dimensional interaction maps in in vitro cultured cell lines. In addition, compared with existing similar technologies, the efficiency is greatly improved. For example, using 2 million cells can obtain about 150 ng of DNA product in step (7).

[0058] Example 3

[0059] (1) Liquid nitrogen quick freezing, grinding and enzymatic hydrolysis of tissue

[0060] 1.1 Take ~50 mg of human prostate cancer tissue or ~50 mg of paracancerous tissue and put it into a mortar. Add 2~3 mL of liquid nitrogen for quick freezing. Use a pestle to grind the quick-frozen tissue blocks. The following operations are for each sample unless otherwise specified.

[0061] 1.2 Transfer the ground tissue into 15 mL of enzymatic solution (100 U / ml collagenase type II; 20 nM ROCK inhibitor Y-27632; 1× antibacterial-antifungal agent; the above components are dissolved in 1× Hank's balanced salt solution) and rotate the enzymatic solution at 37°C for 2 hours;

[0062] 1.3 Centrifuge at 1000g for 5 minutes and wash twice with Hank's balanced salt solution;

[0063] 1.4 Discard the supernatant, add 3 mL of 0.25% TrypLE, and rotate the enzyme for 4 minutes at 37°C;

[0064] 1.5 Add 12 mL of DMEM medium containing 10% FBS to terminate the enzymatic hydrolysis and centrifuge at 1000 g for 5 minutes.

[0065] (2) Cross-linking and cleavage

[0066] 2.1 Centrifuge at 1000g for 5 minutes, wash once with PBS buffer, centrifuge at 1000g for 5 minutes, and discard the supernatant;

[0067] 2.2 Add 10 mL of paraformaldehyde with a final concentration of 1% and cross-link for 10 minutes. During the process, use a tissue homogenizer (Bio-Gen PRO200) to grind 3 times at level 1, each time for 6 seconds;

[0068] 1.3 Add 500 μL of 2.5 M Glycine solution (working concentration 125 mM) and let stand for 5 minutes to terminate cross-linking;

[0069] 1.4 Centrifuge at 1000g for 5 minutes at 4°C, wash once with ice-cold PBS buffer, centrifuge at 1000g for 5 minutes at 4°C, and discard the supernatant;

[0070] 1.5 Add 1 mL of pre-cooled Chromatin Conformation Capture Lysis Buffer (containing 50 mM Hepes–KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5% Igepal CA-630; 0.25% Triton X-100, and protease inhibitors), mix well, and rotate and lyse at 4°C for 50 minutes.

[0071] (3) Enzyme digestion

[0072] 3.1 Centrifuge at 2500g for 5 minutes, discard the supernatant, resuspend in 10mL Chromatin Conformation Capture Lysis Buffer, centrifuge at 2500g for 5 minutes, and discard the supernatant;

[0073] 3.2 Resuspend with 200 μL 0.5% SDS solution at room temperature;

[0074] 3.3 Incubate at 62°C for 15 minutes;

[0075] 3.4 Add 570 μL of triply distilled water and 100 μL of 10% Triton X-100 and mix well;

[0076] 3.5 Incubate at 37°C for 15 minutes;

[0077] 3.6 Add 101.5 μL 10 X NEB buffer 2 and 45 μL (1125 U) restriction enzyme MboI (NEB-R0147);

[0078] 3.7 Enzyme digestion at 37℃ for 2 hours;

[0079] 3.10 Incubate at 70°C for 15 minutes to inactivate MboI.

[0080] (4) Labeling and connection

[0081] 4.1 Add 75 μL 0.4 mM biotin-dATP (Thermo 19524016), 3 μL 10 mM dCTP, 3 μL 10 mM dGTP, 3 μL 10 mM dTTP, and 20 μL 5 U / μL DNA polymerase I (NEB-M0210), and mix well;

[0082] 4.2 Incubate at 37°C for 1 hour;

[0083] 4.3 Add 1896 μL of the prepared ligation solution (containing 300 μL 10X NEB T4 DNA ligase buffer (NEB-B0202), 250 μL 10% Triton X-100, 6 μL 50 mg / mL BSA, 20 μL 400 U / μL T4 DNA ligase (NEB-M0202), and 1320 μL triply distilled water);

[0084] 4.4 Rotate and mix at room temperature for 4 hours;

[0085] 4.5 Centrifuge at 2500g for 5 minutes and discard the supernatant.

[0086] (5) Ultrasound

[0087] 5.1 One day before sonication, mix 40 μL Protein-A with 40 μL Protein-G magnetic beads (Thermofisher, 10002D), wash twice with 0.5% BSA solution (diluted with PBS) on ice, resuspend the magnetic beads with 600 μL 0.5% BSA solution, add 7.5 μg CTCF antibody (CST, 3418S), and rotate overnight at 4°C;

[0088] 5.2 Add 880 μL of ultrasonic buffer (10 mM Tris–HCl, pH 8; 50 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Na–Deoxycholate; 0.5% N-lauroylsarcosine; protease inhibitors added immediately before use) to the ligated product in (4.5), resuspend and mix to a total volume of about 900 μL, and divide evenly into three 1.5 mL ultrasonic tubes;

[0089] 5.3 Perform ultrasound in a pre-cooled Bioruptor® Pico ultrasound machine, with each cycle condition of 10 seconds on + 30 seconds off, repeat 10 cycles, remove the ultrasound tube, shake to mix, and put it back, and continue 10 cycles;

[0090] 5.4 Collect the liquid after sonication into an EP tube, add 90 μL 10% Triton X-100 and mix well;

[0091] 5.5 Centrifuge at 16000g for 15 minutes at 4°C;

[0092] 5.6 Divide the supernatant equally into two EP tubes and add 400 μL of ultrasonic buffer to each tube.

[0093] (6) Chromatin immunoprecipitation

[0094] 6.1 Wash the antibody-magnetic beads pretreated in (5.1) once with 0.5% BSA solution (diluted with PBS) on ice, divide evenly into two EP tubes, precipitate the magnetic beads with a magnetic stand, and discard the supernatant;

[0095] 6.2 Add 400 μL of the product from step 5.6 to each tube of magnetic beads and rotate overnight at 4°C;

[0096] 6.3 On ice, adsorb the magnetic beads using a magnetic stand, discard the supernatant, and resuspend with 1 mL of washing buffer-1 (50 mM Tris, pH 7.6; 150 mM NaCl; 1 mM EDTA; 0.1% SDS; 1% Igepal CA630; protease inhibitors added immediately before use). Repeat the washing process 10 times. For the last wash, collect the magnetic beads of the same sample into one tube.

[0097] 6.4 Wash quickly with 100 mM ammonium bicarbonate solution, adsorb the magnetic beads with a magnetic stand and discard the supernatant.

[0098] (7) DNA extraction

[0099] 7.1 Add 150 μL DNA elution buffer (freshly prepared: 0.1% SDS; 50 mM NaHCO3) and mix well;

[0100] 7.2 Rotate at room temperature for 10 minutes;

[0101] 7.3 Oscillate on a 37°C hot plate for 3 minutes;

[0102] 7.4 Use a magnetic stand to transfer the supernatant to a new tube;

[0103] 7.5 Add 15 μL proteinase K (20 mg / mL);

[0104] 7.6 Oscillate on a 55℃ hot plate for 45 minutes;

[0105] 7.7 67℃ hot plate shaking for 3.5 hours;

[0106] 7.8 DNA was extracted using a DNA purification kit (Zymo). In the last step, 17 μL of triply distilled water was added to elute the DNA, and the elution was repeated once;

[0107] 7.8 Take 1 μL DNA and use Qubit to measure the concentration. The DNA yield is:

[0108] Near cancer: 2.84 ng / μL × 16 μL = 45.44 ng

[0109] Tumor: 4.78 ng / μL × 16 μL = 76.48 ng

[0110] 40 ng of DNA was taken from each sample for subsequent experiments.

[0111] (8) Biotin affinity and transposase cleavage

[0112] 8.1 Take 7.5 μL of Streptavidin-C magnetic beads and wash them once with washing buffer-2 (5 mM Tris-HCl pH 7.5; 0.5 nM EDTA; 1 M NaCl; 0.05% Tween-20);

[0113] 8.2 Add 15 μL 2X biotin binding buffer (20 mM Tris-HCl pH 7.5; 10 mM MgCl2; 20% Dimethylformamide) to the precipitate from step (8.1) and resuspend;

[0114] 8.3 Add 15 μL of DNA obtained in step (7.8) diluted to an appropriate concentration to the magnetic bead suspension in step (8.2);

[0115] 8.4 Rotate or shake at room temperature for 30 minutes;

[0116] 8.5 The magnetic stand absorbs the magnetic beads and discards the supernatant;

[0117] 8.6 Wash the beads twice with 500 μL of Wash Buffer-2, incubating at 55°C with shaking for 2 minutes each time;

[0118] 8.7 Wash the beads once with 100 μL 1X TD buffer;

[0119] 8.8 Add 25 μL 2X TD buffer and 1.3 μL Tn5 transposase, and make up the volume to 50 μL with triply distilled water;

[0120] 8.9 Incubate at 55°C for 10 minutes with interval shaking;

[0121] 8.10 Discard the supernatant, add 500 μL 50 mM EDTA, mix well and shake at 50°C for 30 minutes;

[0122] 8.11 Wash twice with 500 μL 50 mM EDTA, shaking at 50°C for 3 minutes each time;

[0123] 8.12 Wash twice with 500 μL of washing buffer-2, shaking at 55°C for 2 minutes each time;

[0124] 8.13 Wash once with 500 μL 10 mM Tris buffer at room temperature and discard the supernatant.

[0125] (9) Library construction:

[0126] 9.1 First, resuspend the magnetic beads obtained in step (8.13) with 50 μL 1X Phusion High-Fidelity DNA Polymerase Reaction Solution (NEBm0531s);

[0127] 9.2 Use a magnetic rack to discard the supernatant and resuspend the magnetic beads with 50 μL PCR amplification solution (containing 25 μL 2X Phusion high-fidelity DNA polymerase reaction solution, 1 μL 12.5 μM universal amplification primer, 1 μL 12.5 μM barcode-specific amplification primer, and 23 μL triply distilled water);

[0128] 9.3 Preheat the PCR instrument at 72°C in advance, and then perform the PCR amplification program: 72°C 5 minutes -> 98°C 1 minute -> (98°C 15 seconds -> 63°C 30 seconds -> 72°C 1 minute) for 10 cycles;

[0129] 9.4 Use a magnetic rack to transfer the supernatant to a new EP tube. The remaining magnetic beads can be temporarily stored at -20°C. If the first amplification in step 9.3 is unsuccessful, it can be amplified again.

[0130] 9.5 Take 1 μL of PCR product and perform library electrophoresis on Agilent TapeStation to identify the quality of library amplification. Most of the DNA fragments are distributed in the range of 200-700 bp, and the peak is around 400 bp, indicating that the library construction is successful (see Figure 1 );

[0131] 9.6 Use the Pippin DNA Size Selection system to screen 200-700 bp library fragments;

[0132] 9.7 Use NovaSeq (Illumina) platform to perform 150bp paired-end sequencing with a sequencing depth of 150Gb;

[0133] 9.8 Perform quality control and preliminary analysis on the sequencing data. Use hicup software to post reads back to the human genome. The hicup result quality control chart shows that the effective reads of both samples are above 85%, and the data quality is high (see Figure 2 ); Juicer software and R software were used to obtain chromatin three-dimensional interaction loops for APA visualization analysis, showing that the obtained CTCF-mediated chromatin loops were of high strength (the higher the R value, the better the loop quality), and were able to distinguish paracancerous and tumor-specific chromatin loops (see Figure 3 ); The obtained CTCF-mediated chromatin interaction matrix was visualized, showing the typical three-dimensional chromatin interaction characteristics; the above results show that this experiment successfully obtained the CTCF-mediated chromatin three-dimensional interaction map in clinical samples (see Figure 4 ).

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for high-throughput tissue sample chromatin immunoprecipitation combined with chromatin conformation capture for non-diagnostic purposes, characterized in that: The following steps are involved: (1) Tissue liquid nitrogen quick freezing, grinding and enzymatic hydrolysis The tissue was quickly frozen using liquid nitrogen, and then enzymatically hydrolyzed using an enzymatic solution, followed by enzymatic hydrolysis using TrypLE; the enzymatically hydrolyzed cells were then cross-linked using 10 mL of 1% paraformaldehyde, and finally the cross-linking was terminated using a Glycine solution; in step (1), the enzymatic solution was 100 U / ml type II collagenase, 20 nM ROCK inhibitor Y-27632, and 1× antibacterial-antifungal agent, and the above components were dissolved in 1× Hank's balanced salt solution; the enzymatic hydrolysis conditions were rotation enzymatic hydrolysis at 37°C for 2 hours; the enzymatically hydrolyzed cells were cross-linked with paraformaldehyde for 10 minutes, and the tissue homogenizer was used to grind the cells 3 times at level 1, each time for 6 seconds; (2) Tissue lysis Add chromatin conformation capture lysis buffer, which contains 50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5% Igepal CA-630; 0.25% Triton X-100, and protease inhibitors, and rotate and lyse for 50 minutes; (3) Enzyme digestion Tissue lysates were digested with restriction endonuclease MboI; (4) Biotin labeling and ligation Add biotin-dATP, dCTP, dGTP, dTTP, and DNA polymerase to the digested product, label at 37°C for 1 hour; then add ligation solution and ligate at room temperature for 4 hours; (5) Ultrasound One day before sonication, the antibody and immunomagnetic beads are pre-bound, and the connection product obtained in step (4) is resuspended in sonication buffer and sonicated in a sonicator. In step (5), the antibody and immunomagnetic beads are Protein-A and Protein-G magnetic beads, respectively, and the sonication buffer is 10 mM Tris–HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Na–Deoxycholate; 0.5% N-lauroylsarcosine; protease inhibitors are added immediately after use; the sonication condition is 10 seconds on + 30 seconds off for each cycle, and 10 cycles are repeated. The sonication tube is taken out and shaken to mix well, and then put back, and another 10 cycles are continued. (6) Chromatin immunoprecipitation The antibody-magnetic beads pretreated in step (5) were mixed with the ultrasonic product and rotated overnight at 4°C; after overnight, the magnetic beads were washed with a washing buffer solution and an ammonium bicarbonate solution respectively; (7) DNA extraction Elute the DNA on the magnetic beads using a DNA elution buffer, and then extract the DNA using a DNA purification kit; (8) Biotin affinity and transposase cleavage Resuspend the Streptavidin-C magnetic beads in reaction buffer, and add the DNA solution obtained in step (7) to the Streptavidin-C magnetic bead suspension, and perform affinity at room temperature for 30 minutes; add reaction buffer and Tn5 transposase to the Streptavidin-C magnetic beads bound to the biotin-labeled DNA, and cut at 55°C for 10 minutes with interval shaking; (9) Library construction First, use Phusion high-fidelity DNA polymerase reaction solution to pre-clean the Streptavidin-C magnetic beads obtained in step (8), and use PCR amplification solution to amplify the DNA on the Streptavidin-C magnetic beads. Set the PCR instrument to 72°C in advance to ensure that the reaction solution heats up quickly. The PCR amplification program is: 72°C 5 minutes -> 98°C 1 minute -> 98°C 15 seconds -> 63°C 30 seconds -> 72°C 1 minute, 9 to 12 cycles; The PCR products were subjected to library electrophoresis on Agilent TapeStation to identify the quality of library amplification; the 200-700 bp library fragments were screened using the Pippin DNA Size Selection system or Ampure XP magnetic beads.

2. A method for high-throughput tissue sample chromatin immunoprecipitation combined with chromatin conformation capture for non-diagnostic purposes according to claim 1, characterized in that: In the step (3), the restriction endonuclease MboI digestion condition is 37°C for 2 hours.

3. A method for high-throughput tissue sample chromatin immunoprecipitation combined with chromatin conformation capture for non-diagnostic purposes according to claim 1, characterized in that: In step (4), the ligation solution contains 300 μL 10X NEB T4 DNA ligase buffer, 250 μL 10% Triton X-100, 6 μL 50 mg / mL BSA, 20 μL 400 U / μL T4 DNA ligase, and 1320 μL triply distilled water.

4. A method for high-throughput tissue sample chromatin immunoprecipitation combined with chromatin conformation capture for non-diagnostic purposes according to claim 1, characterized in that: In step (6), the washing buffer is 50 mM Tris, pH 7.6; 150 mM NaCl; 1 mM EDTA; 0.1% SDS; 1% Igepal CA630; 0.5% Na-Deoxycholate; and protease inhibitors are added immediately after use.

5. The method of high-throughput tissue sample chromatin immunoprecipitation combined with chromatin conformation capture for non-diagnostic purposes according to claim 1, characterized in that: In step (7), the elution step is as follows: rotate at room temperature for 10 minutes, shake on a hot plate at 37°C for 3 minutes, transfer the supernatant to a new tube, add proteinase K, shake on a hot plate at 55°C for 45 minutes, and shake on a hot plate at 67°C for 3.5 hours.

6. A method for high-throughput tissue sample chromatin immunoprecipitation combined with chromatin conformation capture for non-diagnostic purposes according to claim 1, characterized in that: In the step (9), the PCR amplification solution contains 25 μL 2X Phusion high-fidelity DNA polymerase reaction solution, 1 μL 12.5 μM universal amplification primer, 1 μL 12.5 μM barcode-specific amplification primer, and 23 μL triply distilled water.

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