Small-amount efficient specific factor mediated whole genome chromatin interaction detection technology Solid ChIA-PET

Abstract

The invention relates to a whole genome chromatin interaction detection technology Solid ChIA-PET mediated by a small amount of efficient specific factors. The technology relates to a sample pretreatment method, and the sample pretreatment method comprises the steps that cells are attached to the surface of a treated solid, and on the surface of the solid, the operation steps of cell cross-linking fixation, cell lysis, cell nucleus lysis and the like can be sequentially completed. Subsequently, the target chromatin region is recognized using a specific antibody, and the target chromatin region is cleaved by protein G-micrococcus nuclease, followed by a proximity ligation step. And further cutting the chromatin fragment through a Tn5 transposase reaction, and integrating a sequencing joint. The target protein mediated whole genome chromatin interaction detection method comprises the step of treating a sample by adopting the sample pretreatment method, and the method breaks through the limitation of a traditional method in treating a small number of cell samples and is compatible with a single cell experiment.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to a genome-wide chromatin interaction detection technology mediated by a small amount of highly efficient specific factors - Solid ChIA-PET. Background Art

[0002] Linear genomic sequences are highly folded in the cell nucleus, forming a complex three-dimensional structure. This spatial organization allows gene transcription to be regulated by remote regions, providing evidence for long-range regulation that leads to disease in many noncoding region mutations discovered during whole-genome sequencing. This regulation is often associated with transcription factors. Currently, the classic method for examining the spatial structure of chromatin mediated by specific protein factors is mainly ChIA-PET technology. This technology is used to analyze chromatin interaction networks mediated by chromatin organizing factors (such as CTCF) and transcription factors (such as RNAPII), and their effects on chromatin spatial structure and gene transcription. Although ChIA-PET technology has made significant progress in elucidating the mechanisms of gene transcription regulation, it requires a large number of cells (millions or even billions) to obtain high-quality maps, making it difficult to apply to clinical samples to solve practical problems. Summary of the Invention

[0003] Based on this, one embodiment of the present application provides a small amount of efficient specific factor-mediated whole-genome chromatin interaction detection technology_Solid ChIA-PET and a target protein-mediated whole-genome chromatin interaction detection method based on this technology. This detection method is efficient and suitable for a small amount of samples.

[0004] The technical solution includes:

[0005] A sample pre-treatment method, comprising the following steps:

[0006] 1) Cross-linking and fixing the cell or tissue sample, and then performing cell lysis and cell nucleus lysis on the cross-linked and fixed sample;

[0007] 2) incubating the obtained cell nuclei with a specific antibody for the target protein to prepare a cell nucleus bound by the specific antibody; wherein the target protein is located on chromatin in the cell nucleus;

[0008] 3) using protein G-micrococcal nuclease to fragment the chromatin in the cell nucleus bound by the heterologous antibody in situ;

[0009] 4) perform proximity ligation on fragmented chromatin; and

[0010] 5) Using Tn5 transposase to in situ cut the ligated chromatin and insert a sequencing adapter into the cut site to prepare a cell nucleus containing chromatin with sequencing adapters;

[0011] Here, before cross-linking and fixation, the method further includes a step of reversibly attaching the cell or tissue sample to a solid surface, or, before incubating the cell nucleus with a specific antibody of the target protein, further includes a step of reversibly attaching the cell nucleus to a solid surface.

[0012] In one embodiment, the solid surface is a solid surface treated with poly-lysine.

[0013] In one embodiment, in step 3), Ca 2+ Activation of the protein G-micrococcal nuclease results in in situ fragmentation of chromatin.

[0014] In one embodiment, in step 4), the fragmented chromatin DNA is blunt-ended with T4 DNA polymerase, the blunt-end-repaired DNA ends are A-tailed with Klenow fragment 3'-5' exo-, and the chromatin DNA is proximity-ligated with a biotin-labeled and T-tail-modified oligonucleotide.

[0015] In one embodiment, in step 1), the fixative used for cross-linking fixation includes formaldehyde.

[0016] In one embodiment, in step 1), one or both of the following conditions are met:

[0017] (a) lysing the cells by vortexing using a cell lysis buffer, wherein the cell lysis buffer comprises 0.1% w / v SDS;

[0018] (b) vortexing the resulting solution with a nuclear lysis buffer to lyse the cell nuclei; wherein the nuclear lysis buffer comprises 1% w / v SDS;

[0019] Optionally, the temperature for the rotation mixing is room temperature, and the time for the rotation mixing is 10 min-25 min.

[0020] The method for detecting genome-wide chromatin interactions mediated by target proteins includes the following steps:

[0021] Perform sample pretreatment using the method described; and

[0022] The processed samples were used for library construction and high-throughput sequencing.

[0023] In one embodiment, library construction and high-throughput sequencing include the following steps:

[0024] Reversing cross-linking of the obtained cell nuclei including chromatin with sequencing adapters, purifying DNA, and enriching DNA containing biotin labeling; and

[0025] The enriched DNA was amplified by PCR, fragment screened, and subjected to high-throughput sequencing.

[0026] In one embodiment, the cell nuclei including the chromatin with sequencing adapters are decrosslinked using proteinase K at 55°C-65°C.

[0027] A genome-wide chromatin interaction detection method for target proteins in single cells includes the following steps:

[0028] Performing sample pretreatment using the method; and

[0029] Single cell nuclei suspensions were prepared from the treated samples.

[0030] Optionally, a cell sorter is used to construct a low-throughput single-cell labeling library for the single-cell nucleus suspension, or a high-throughput single-cell labeling library is constructed based on microfluidics technology.

[0031] Optionally, the cell nuclei including the chromatin with sequencing adapters are separated from the solid surface using trypsin to prepare a single cell nucleus suspension.

[0032] Compared with traditional technology, this application has the following beneficial effects

[0033] 1. Introducing a solid-phase environment to optimize experimental operations: Traditional liquid-phase operations have significant limitations when processing small amounts of cell samples. For example, when the number of cells is less than 100,000, the cell pellet is not visible after centrifugation, making liquid replacement and resuspension difficult, and even causing sample loss. This application constructs a solid-phase environment by attaching cells or treated cell nuclei to a treated solid surface (such as a glass slide or well plate), thereby significantly optimizing experimental operations. In this solid-phase environment, the buffer or reaction system can be directly replaced, avoiding the tedious steps of frequent centrifugation and resuspension in traditional methods, thereby improving the convenience of operation and the stability of the experiment. By fixing the cell nuclei on the solid phase, this application can process samples as low as 200 cells, which is particularly suitable for small amounts of cell samples (such as clinical samples and rare cell populations), greatly improving the operability of small amounts of samples.

[0034] 2. Separable adhesion strategy to achieve efficient single-cell nuclear suspension preparation: A separable adhesion strategy is used to allow cells to be firmly attached to the solid surface during the experiment, and after the experiment, the cell nuclei are separated from the solid surface by physical separation (such as blowing with a pipette), thereby recovering the cell nuclei and preparing them into single-cell nuclear suspensions, with a recovery rate of more than 95%. Compared with the situation in which cells agglomerate due to multiple centrifugation and fluid changes in traditional liquid phase operations, this strategy not only simplifies the experimental process and improves the convenience of operation, but also ensures that even a small number of cells can be successfully prepared into single-cell nuclear suspensions after multiple rounds of fluid changes. The present application provides an efficient and stable single-cell nuclear suspension preparation solution with a higher recovery rate and better operability.

[0035] 3. An innovative "targeted cleavage followed by ligation" strategy improves detection sensitivity and specificity: The "targeted cleavage followed by ligation" strategy uses specific antibodies to identify and locate the target protein-mediated chromatin region. It then uses pG-MNase to cleave the DNA on either side of the target protein, followed by a proximity ligation reaction. Compared to the "ligation followed by targeted cleavage" strategy of Hi-Tag and HiCuT, this approach avoids the problem of linker-containing fragments being destroyed during the cleavage process, ensuring accurate cleavage of the target protein-mediated chromatin region and labeling with linkers through proximity ligation, significantly improving the sensitivity and specificity of chromatin interaction detection.

[0036] 4. Tn5 transposase in situ transposition captures multi-dimensional information: After proximity ligation, Tn5 transposase cleaves the chromatin fragments in situ and integrates sequencing adapters. This process captures not only target protein-mediated chromatin interactions but also information about accessible chromatin regions.

[0037] The method in this application is simple and efficient to operate, and is suitable for small amounts of samples. It is expected to provide a powerful tool for detecting chromatin interactions in clinical specimens, and provide broader application prospects for studying complex chromatin interaction networks, especially in the study of clinical samples and rare cell populations. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0039] Figure 1 Schematic diagram of the Solid ChIA-PET technical solution.

[0040] Figure 2 Schematic diagram of the detailed experimental process of Solid ChIA-PET.

[0041] Figure 3 is the sequence of the bridge linker.

[0042] Figure 4 The size distribution of DNA fragments in open chromatin regions was obtained using Solid ChIA-PET technology.

[0043] Figure 5 The following are the microscopic examination results of different initial cell numbers (200, 2,000, and 20,000) after multiple rounds of reaction on the well plate for 3 days.

[0044] Figure 6 The cells were separated from the solid surface and the single cell nucleus suspension was prepared for microscopic examination.

[0045] Figure 7 Signal comparison of target protein enrichment regions and target protein-mediated chromatin interactions using Solid ChIA-PET, in situ ChIA-PET, and Solution ChIA-PET methods.

[0046] Figure 8 Visual display of target protein enrichment areas, target protein-mediated chromatin interactions, and chromatin open areas using the Solid ChIA-PET method. DETAILED DESCRIPTION

[0047] To make the above-mentioned objects, features, and advantages of the present application more clearly understood, the specific embodiments of the present application are described in detail below. The following description sets forth many specific details to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without violating the scope of the present application. Therefore, the present application is not limited to the specific embodiments disclosed below.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0049] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0050] Traditional ChIA-PET technology uses specific antibodies to capture target proteins (such as transcription factors or chromatin organizing factors) in chromatin complexes, and then uses biotin-labeled adapters to connect DNA fragments in the chromatin complexes. Through high-throughput sequencing, ChIA-PET technology can identify DNA fragments that interact remotely in chromatin, revealing the three-dimensional structure of chromatin and the gene regulatory network. ChIA-PET can not only identify DNA sites bound by specific proteins, but also provide spatial interaction information between these sites. This gives ChIA-PET a unique advantage in analyzing the complex three-dimensional structure of chromatin and understanding gene regulatory mechanisms. However, traditional ChIA-PET technology captures DNA and protein complexes through chromatin immunoprecipitation, and usually requires a large number of cell samples to obtain high-quality interaction maps, which to some extent limits its application in clinical samples and rare cell populations.

[0051] One embodiment of the present application provides a sample pre-treatment method, which is suitable for sample pre-treatment for target protein-mediated whole-genome chromatin interaction detection, comprising the following steps 1) to 5):

[0052] 1) Cross-link and fix the cell or tissue sample, and then perform cell lysis and cell nucleus lysis on the cross-linked and fixed sample.

[0053] In a specific example, before cross-linking and fixation, a step of reversibly attaching the cell or tissue sample to a solid surface is further included.

[0054] In one embodiment, the solid surface is a solid surface treated with poly-lysine.

[0055] In one specific example, the solid body comprises a well plate or a glass slide.

[0056] In one specific example, a fixative is used for cross-linking fixation.

[0057] In a specific example, the well plate or slide is treated with polylysine, left to stand at room temperature for 20 min-35 min, the polylysine is removed, and the well plate or slide is allowed to dry.

[0058] In one embodiment, cells are seeded (attached to or anchored to) the treated solid surface by centrifugation.

[0059] In a specific example, the fixing agent includes formaldehyde. Optionally, the fixing agent includes a 30 wt %-40 wt % formaldehyde solution, and optionally a 36 wt % formaldehyde solution.

[0060] In a specific example, the cross-linking fixation conditions include fixation at room temperature for 5 min-15 min, optionally 10 min.

[0061] In a specific example, the cell sample or tissue sample is fixed with a 36 wt % formaldehyde solution at room temperature for 10 minutes to terminate the cross-linking reaction, and the formaldehyde is removed by washing with DPBS, and the cell pellet is collected.

[0062] In one embodiment, the cell lysis solution used for cell lysis includes 0.1% w / v SDS.

[0063] In a specific example, cell lysis includes resuspending the cell pellet with a lysis buffer and rotating the cell pellet at room temperature for 10 min to 25 min.

[0064] In one specific example, the nuclear lysis solution used for cell nuclei lysis includes 1% w / v SDS. The lysate after cell lysis is mixed with a nuclear lysis solution containing 1% w / v SDS at room temperature for 5-15 minutes with rotation, and the nuclei are collected. The purpose of nuclear lysis is primarily to disrupt the cell membrane and loosen the chromatin.

[0065] 2) Incubating the obtained cell nuclei with a specific antibody for the target protein, wherein the target protein is located on chromatin in the cell nucleus, to prepare a cell nucleus bound by the specific antibody;

[0066] In a specific example, before incubating the cell nuclei with the specific antibody of the target protein, the method further includes a step of reversibly attaching the cell nuclei to a solid surface.

[0067] In one embodiment, the solid surface is as described above.

[0068] In one embodiment, the cell nuclei are seeded (attached) to the treated solid surface by centrifugation.

[0069] In a specific example, the cell nuclei are incubated with a 0.05% w / v SDS lysis buffer containing a specific antibody against the target protein, so that the target protein on the chromatin in the cell nucleus is bound in situ by the specific antibody.

[0070] In one embodiment, after incubation, the excess unbound specific antibody is removed.

[0071] 3) In situ fragmentation of chromatin in the nuclei bound by heterologous antibodies was performed using protein G-micrococcal nuclease.

[0072] In one embodiment, protein G-micrococcal nuclease is incubated with cell nuclei bound to a heterologous antibody to activate protein G-micrococcal nuclease and fragment chromatin in situ.

[0073] In a specific example, using Ca 2+ Activating protein G-micrococcal nuclease.

[0074] 4) Perform proximity ligation on fragmented chromatin.

[0075] In a specific example, the fragmented chromatin DNA is blunt-ended using T4 DNA polymerase, the blunt-end repaired DNA ends are A-tailed using Klenow fragment (3'-5'exo-), and the chromatin DNA is proximity-ligated using a biotin-labeled and T-tail-modified oligonucleotide.

[0076] In a specific example, the concentration of T4 DNA polymerase in the reaction system is about 0.05U / µL-0.1U / µL.

[0077] In a specific example, blunt-end repair is performed at 36° C.-38° C. with shaking at 10 rpm-15 rpm for 30 min-40 min.

[0078] In a specific example, the concentration of Klenow fragment (3'-5'exo-) in the reaction system is 0.05-0.1 U / µL.

[0079] In a specific example, the DNA ends after blunt end repair are A-tailed at 36° C.-38° C. and shaken at 10 rpm-15 rpm for 40 min-60 min.

[0080] In a specific example, for 1M cells, the amount of oligonucleotide sequence (Bridge linker) added is 30 ng.

[0081] In a specific example, the oligonucleotide includes the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, wherein the deoxythymidylate T at position 10 of SEQ ID NO: 1 is modified with biotin.

[0082] 5) Using Tn5 transposase to in situ cut the ligated chromatin and insert sequencing adapters into the cut sites to prepare cell nuclei including chromatin with sequencing adapters.

[0083] Cell nuclei, including chromatin with sequencing adapters, can be used for library construction and high-throughput sequencing, or to prepare single-cell nuclear suspensions for subsequent single-cell experiments.

[0084] It should be noted that, according to the high-throughput sequencing method, a sequencing adapter suitable for the sequencing platform is selected.

[0085] One embodiment of the present application further provides a method for detecting genome-wide chromatin interactions mediated by a target protein, comprising the following steps:

[0086] performing sample pretreatment using the sample pretreatment method; and

[0087] The processed samples were used for library construction and high-throughput sequencing.

[0088] In a specific example, library construction and high-throughput sequencing include the following steps:

[0089] Reversing cross-linking of the obtained cell nuclei including chromatin with sequencing adapters, purifying DNA, and enriching DNA containing biotin labeling; and

[0090] The enriched DNA was amplified by PCR, fragment screened, and subjected to high-throughput sequencing.

[0091] In one specific example, fragments between 300 bp and 600 bp were screened.

[0092] In one embodiment, the cell nuclei including the chromatin with sequencing adapters are decrosslinked using proteinase K at 55°C-65°C.

[0093] One embodiment of the present application further provides a method for detecting genome-wide chromatin interactions mediated by a target protein in a single cell, comprising the following steps:

[0094] performing sample pretreatment using the sample pretreatment method; and

[0095] Use the treated samples to prepare single-cell nuclear suspensions, perform single-cell experiments on the single-cell suspensions, and investigate chromatin interactions mediated by target proteins at the single-cell level.

[0096] In one embodiment, cell nuclei including chromatin with sequencing adapters are detached from a solid surface using trypsin to prepare a single cell nucleus suspension.

[0097] This application provides a highly efficient, genome-wide chromatin interaction detection method (named Solid ChIA-PET) mediated by target proteins (such as transcription factors or chromatin organizing factors) suitable for small sample sizes. This technology involves attaching cells to a treated solid surface, where steps such as cross-linking, fixation, and lysis can be performed sequentially. Alternatively, the cross-linked, fixed cells can be lysed and treated with cell and nuclear lysis buffers. The cells are then attached to the solid surface, enabling population-level experimental manipulation of small numbers of cells and tissue samples. Furthermore, cells attached to the solid surface can be physically recovered for subsequent single-cell experiments, thus addressing both population and single-cell research needs. Subsequently, specific antibodies are used to identify the target chromatin regions, which are then cleaved using pG MNase, followed by a proximity ligation step. Next, the chromatin fragments are further cleaved using a Tn5 transposase reaction, and sequencing adapters are incorporated. The ligation products are captured and sequenced, ultimately yielding the chromatin interaction network mediated by the specific antibodies.

[0098] This method overcomes the limitations of traditional methods when processing small cell samples and is also compatible with single-cell experiments. By first using specific antibodies to mediate chromatin cleavage and then performing proximity ligation at the cleavage sites, the efficiency of capturing chromatin interactions and ligation efficiency are significantly improved, as well as the sensitivity, specificity, and accuracy of chromatin interaction detection. In addition, this method can also provide information on open chromatin regions, providing a powerful tool for analyzing the three-dimensional structure and regulatory networks of the genome.

[0099] One embodiment of the present application also provides a kit for detecting whole-genome chromatin interactions mediated by a target protein, the kit comprising protein G-micrococcal nuclease and Tn5 transposase, and also comprising a sequencing adapter, a lysis buffer, a solid, T4 DNA polymerase, Klenow Fragment (3'-5' exo-), Ca 2+ , one or more of a biotin-labeled and T-tail-modified oligonucleotide sequence, a fixative, streptavidin magnetic beads, and a PCR amplification reagent.

[0100] In one embodiment, the lysis buffer comprises 0.1% w / v SDS, which is suitable for lysing cell membranes.

[0101] In one embodiment, the lysis buffer comprises 1% w / v SDS and is suitable for lysing cell nuclei.

[0102] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0103] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0104] like Figure 1As shown, Solid ChIA-PET is a highly efficient, genome-wide technique for detecting chromatin interactions mediated by target proteins using small amounts of target proteins. First, cross-linked, fixed cells are lysed and treated with cell lysis buffer and nuclear lysis buffer. The cells are then adhered to a solid surface (such as a well plate or slide), facilitating manipulation of small cell or tissue samples. Next, the cells are incubated with specific antibodies and fragmented in situ using pG MNase. The cleaved chromatin DNA ends are then ligated with biotinylated adapters. The adhered cells can then be physically separated by trypsin treatment and recovered for single-cell experiments. Alternatively, proteins can be digested with a protease, and the ligated DNA can be extracted and purified. The extracted DNA fragments can be further enriched for adapter-containing fragments using streptavidin magnetic beads and amplified by PCR to construct a population-level Solid ChIA-PET library. Solid ChIA-PET is suitable for profiling and mapping chromatin interactions in small numbers of cells, or even single cells.

[0105] Example 1

[0106] Taking 100,000 Drosophila Schneider 2 (S2) cells and RNA polymerase II as the target protein as an example, the whole genome chromatin interaction detection method (Solid ChIA-PET) of this application is described. The method includes the following steps (see Figure 2 ):

[0107] (1) Cross-linking and fixation of cell or tissue samples: 400 million Drosophila Schneider 2 (S2) cells were centrifuged to remove the culture medium and resuspended in 40 mL DPBS (Thermo Fisher, catalog number 14190250). 1.1 mL 36 wt% formaldehyde (Sigma, catalog number 47608) was added and rotated at room temperature for 10 minutes. Subsequently, 3.6 mL 2.5 M glycine solution (Sigma, catalog number 50046) was added to the cell suspension to make the final glycine concentration reach 0.2 M. The cross-linking reaction was terminated by rotating at room temperature for 10 minutes. The cells were then centrifuged at 2500 rpm for 5 minutes at room temperature to collect the cell pellet. After removing the supernatant, the cells were resuspended in 40 mL DPBS and rotated at room temperature for 5 minutes. The cells were centrifuged again at 2500 rpm for 5 minutes and the supernatant was discarded. The washing step was repeated three times to ensure the removal of formaldehyde. After fixation, cells can be aliquoted into tubes of 2 million cells each and stored at -80°C for subsequent use. To prepare a 2.5 M glycine solution, dissolve 9.375 g of glycine in 40 mL of deionized water and stir until the solution is clear. Then, bring the final volume to 50 mL with deionized water. The solution can be stored at room temperature for 6 months.

[0108] (2) Take out the cell or tissue sample stored at -80℃, thaw it on ice, resuspend it in DPBS, centrifuge it at 5000rpm for 5 minutes at room temperature, remove the supernatant, and then resuspend the cells in 1mL of cell lysis buffer containing protease inhibitors and rotate at room temperature for 20 minutes to fully lyse. The cell lysis buffer is prepared by adding 1.25mL 1M Tris-HCl pH7.0 (Thermo Fisher, catalog number AM9851), 1.25mL 1M Tris-HCl pH 8.0 (Thermo Fisher, catalog number AM9856), 1.5mL 5M NaCl (Thermo Fisher, catalog number AM9759), 100µL 0.5M EDTA (Thermo Fisher, catalog number AM9260G) and 0.5mL 10% w / v SDS (Thermo Fisher, catalog number AM9822), and add deionized water to a final volume of 50mL. It can be stored at room temperature for 6 months. Prepare protease inhibitor stock solution (50× concentration): Add one cOmplete EDTA-free protease inhibitor tablet (Roche, catalog number 11873580001) to 1 mL of deionized water and rotate at 4°C until completely dissolved. Prepare 1 mL of cell lysis buffer containing protease inhibitors by adding 20 µL of protease inhibitor stock solution (50× concentration) to 980 µL of cell lysate on the day of the experiment.

[0109] (3) Nuclear lysis: Add 100 µL of 10% w / v SDS to 1 mL of cell lysis buffer, mix well, and rotate at room temperature for 5 minutes. Centrifuge at 5000 rpm for 5 minutes at room temperature and discard the supernatant. Observe the cell lysis under a microscope. Repeat the above nuclear lysis steps if necessary until nuclear lysis is complete.

[0110] (4) Resuspend the cell pellet in 1 mL of DPBS solution, wash once, centrifuge at 5000 rpm for 5 minutes at room temperature, and discard the supernatant. Then resuspend the cell pellet in 1 mL of TritonX-100 aqueous solution and rotate at 37°C for 30 minutes to remove excess SDS. Subsequently, centrifuge at 5000 rpm for 5 minutes at room temperature, discard the supernatant, and resuspend the cell pellet in DPBS solution before adding it to the well plate. The TritonX-100 aqueous solution is prepared by adding 50 µL of 20% w / v TritonX-100 (Acros Organics, catalog number 327371000) and 20 µL of protease inhibitor stock solution (50× concentration) and making up to a final volume of 1 mL with DPBS.

[0111] (5) Antibody incubation: Treat the well plate or slide with 0.01% w / v poly-lysine solution (Sigma, catalog number P4707), remove the poly-lysine after standing at room temperature for 30 minutes, and let it dry. Rinse the well plate once with DPBS, dry it again, add 100,000 prepared cells to the well plate, centrifuge at 3500 rpm for 20 minutes at room temperature, then gently change the solution and observe under a microscope whether the cells are firmly attached to the well plate surface. Tilt the well plate to discard the supernatant, add 500µL of antibody buffer containing protease inhibitors to each well, and incubate at 4°C overnight to allow chromatin proteins to bind to specific antibodies in situ. Prepare antibody buffer by adding 1.25 mL of 1M Tris-HCl (pH 7.0), 1.25 mL of 1M Tris-HCl (pH 8.0), 1.5 mL of 5M NaCl, 100 µL of 0.5M EDTA, 0.25 mL of 10% w / v SDS, and 2.5 mL of 20% w / v Triton X-100. Bring the volume up to 50 mL with deionized water. Prepare antibody buffer containing protease inhibitors by adding 10 µL of protease inhibitor stock solution (50× concentration) and 10 µL of 1 µg / µL RNA polymerase II antibody (BioLegend, Cat. No. 664906) to 490 µL of antibody buffer for a 1:50 dilution.

[0112] (6) In situ fragmentation with pG MNase (Protein G-micrococcal nuclease): After overnight incubation with antibodies, the plate was tilted to discard the supernatant, 500µL of low-salt buffer containing protease inhibitors was added, and the plate was shaken at 10 rpm for 5 minutes at room temperature. The buffer was then discarded, and the same washing steps were performed once with high-salt buffer, DPBS solution, and Dig-wash buffer in sequence to remove excess primary antibody. Subsequently, 1µL of 100-fold diluted pG MNase (Norvozymes, catalog number HD101) was mixed with 100µL Dig-wash buffer and added to the plate. The cells were incubated with the antibody-bound cells at 4°C for 1.5 hours to allow pGMNase to bind to the specific antibody. After the incubation, the supernatant was discarded, 500µL of Dig-wash buffer was added, and the plate was shaken at 10 rpm for 5 minutes at room temperature to wash away excess nuclease and discard the supernatant again. Subsequently, 2 µL of CaCl₂ (Novagen, catalog number HD101) was added to 98 µL of Dig-wash buffer, mixed thoroughly, and added to the plate. The plate was incubated at 0°C for 1.5 hours to initiate the pG MNase fragmentation reaction. Following the digestion reaction, 100 µL of Dig-wash buffer containing 0.02 M EDTA was added, and the plate was shaken at 10 rpm for 5 minutes at room temperature before the buffer was discarded to terminate the digestion reaction. Low-salt buffer was prepared by adding 1.25 mL of 1 M Tris-HCl (pH 7.0), 1.25 mL of 1 M Tris-HCl (pH 8.0), 1.5 mL of 5 M NaCl, 100 µL of 0.5 M EDTA, 0.5 mL of 10% w / v SDS, and 2.5 mL of 20% w / v Triton X-100. The final volume was made up to 50 mL with deionized water. The plate can be stored at 4°C for 6 months. Before use, add protease inhibitors to a final concentration of 1x. Prepare high-salt buffer by adding 1.25 mL of 1M Tris-HCl (pH 7.0), 1.25 mL of 1M Tris-HCl (pH 8.0), 3.5 mL of 5M NaCl, 100 µL of 0.5M EDTA, 0.5 mL of 10% w / v SDS, and 2.5 mL of 20% w / v Triton X-100. Bring the volume up to 50 mL with deionized water. Store at 4°C for 6 months. Before use, add protease inhibitors to a final concentration of 1x. For Dig-wash buffer, refer to the instructions for the Novozymes HD101 kit: add 160 µL of 10x wash buffer and 32 µL of 50x protease inhibitor stock solution. Bring the volume up to 1.6 mL with deionized water. Mix thoroughly, then add 16 µL of 5% w / v Digitonin.

[0113] (7) Proximity ligation: Tilt the plate to discard the supernatant, add 200 µL T4 DNA polymerase mixture (1×T4 DNA Polymerase buffer (Promega, catalog number 0000629469), 100 µM dNTPs (NEB, catalog number N0447VVIAL), 0.05 U / µL T4 DNA Polymerase (Promega, catalog number M4211)), shake at 10 rpm at 37°C for 40 minutes to blunt-end the chromatin fragments after nuclease cutting. Next, the plate was tilted to discard the supernatant, and 500 µL of a Klenow fragment 3'-5'exo-mix (1× NEB rCutsmart buffer (NEB, catalog no. B6004SVIAL), 0.4 mg / mL BSA (TaKaRa, catalog no. 2320), 0.04 mM dATP (ThermoFisher, catalog no. 18252015), 0.5% w / v Triton X-100, and 0.05 U / µL Klenow fragment 3'-5'exo- (NEB, catalog no. M0212L)) was added. The reaction was shaken at 10 rpm at 37°C for 1 hour to add A tails to the 3' ends of the blunt-ended chromatin DNA. After completion of the reaction, the cells were washed three times with ChIA-PET Wash buffer. Subsequently, the plate was tilted to discard the supernatant, and 500 μL of T4 DNA ligase mixture (1×T4 DNA ligase buffer (NEB, catalog number M0202SVIAL), Bridge linker, 4 U / μL T4 DNA ligase (NEB, catalog number M0202S)) was added. The mixture was shaken at 10 rpm for 1 hour at room temperature, and then transferred to 16°C for overnight reaction to perform "proximity ligation" on the chromatin. For 100,000 S2 cells, the amount of Bridge linker added was 3 ng. Bridge linker is an 18 bp double-stranded DNA oligonucleotide sequence with a biotin label and T-tail modification. The sequence is as follows: Figure 3The bridge linker consists of two strands, one of which (SEQ ID NO:1) has an oligonucleotide sequence of 5'-CGCGATATCTTATCTGACT-3', and the other (SEQ ID NO:2) has an oligonucleotide sequence of 5'-GTCAGATAAGATATCGCGT-3'. The deoxythymidylate (T) at position 10 in SEQ ID NO:1 is biotinylated. In proximity ligation, the "T" overhang of the bridge linker can pair with the "A" overhang formed by A-tailed chromatin fragments, thereby improving proximity ligation efficiency. ChIA-PET Wash buffer is prepared by adding 250µL of 1M Tris-HCl (pH 7.0), 250µL of 1M Tris-HCl (pH 8.0), 1.5mL of 5M NaCl, and 100µL of 0.5M EDTA, and bringing the volume to a final volume of 50mL with deionized water. It can be stored at 4°C for 6 months. Upon use, add protease inhibitors to a final concentration of 1x.

[0114] (8) In situ tagmentation with Tn5 transposase: Tilt the plate to discard the supernatant, add 200µL ATAC Wash buffer to wash once, then add 150µL Tn5 transposase mixture and incubate at 55°C for 10 minutes to allow Tn5 transposase to cut the chromatin region in situ and integrate the sequencing adapter into the chromatin fragment at the cutting site. ATAC Wash buffer consists of 10mM Tris-HCl (pH 7.4), 10mM NaCl, 3mM MgCl2 and 0.1% w / v Tween-20 (Sigma, catalog number P1379). It must be prepared fresh before use and kept on ice. To prepare the Tn5 transposase mix, add 37.5 µL of 4× THS TD buffer and 6 µL of Tn5 transposase (BGI Genomics, catalog number LS-EZ-E-000090). Add deionized water to a final volume of 150 µL, mix thoroughly, and place on ice. The amount of Tn5 transposase used should be adjusted based on the number of cells used. Prepare 4× THS TD buffer by adding 132 µL of 1M Tris-HCl (pH 8.0), 52.8 µL of 5M potassium acetate (Sigma Aldrich, catalog number 95843-100ML-F), 40 µL of 1M magnesium acetate (Sigma Aldrich, catalog number 63052-100ML), and 640 µL of 100% N,N-dimethylformamide (Sigma Aldrich, catalog number 227056-100ML). Bring to a final volume of 1 mL with deionized water. Store at -20°C for 2 months.

[0115] (9) Decrosslinking and DNA purification: After in situ labeling by Tn5 transposase, immediately add 95µL pH 8.0 TE buffer (Thermo Fisher, catalog number AM9849), 250µL ChIP Elution buffer and 5µL 20mg / mL proteinase K (Thermo Fisher, catalog number AM2548) and shake at 10rpm at 37℃ overnight. Proteinase K will terminate the in situ labeling reaction of transposase and digest the protein components in the complex, while decrosslinking the chromatin complex. After decrosslinking, observe whether the cells are completely lysed under a microscope, collect the solution in the well plate into a 1.5mL centrifuge tube, and then use the phenol-chloroform method to extract DNA. Add 500µL phenol:chloroform:isoamyl alcohol (25:24:1) (Solarbio, catalog number P1012) to the solution, invert it and centrifuge it at 12,000rpm for 6 minutes at room temperature, and transfer the supernatant to a 1.5mL centrifuge tube. Next, add 52 µL of 3M sodium acetate (Thermo Fisher, catalog number AM9740), 1 µL of 15 mg / mL GlycoBlue coprecipitant (ThermoFisher, catalog number AM9516), and 520 µL of isopropanol (Sigma, catalog number I9030). Mix thoroughly by gentle inversion and store at -80°C overnight. The next day, remove the mixture from -80°C and thaw at room temperature. Centrifuge at 12,000 rpm for 1 hour at 4°C to precipitate the DNA. Remove the supernatant and add 800 µL of ice-cold 75% ethanol (Sigma, catalog number E7023-500ML). Gently shake the tube to resuspend the pellet, then centrifuge at 12,000 rpm for 5 minutes at 4°C and remove the supernatant. Repeat this wash process once more. Finally, vacuum-dry the DNA pellet and elute the DNA with 100 µL of EB buffer (Qiagen, catalog number 19086) by incubating at room temperature for 1 to 2 hours. Prepare the ChIA-PET elution buffer by adding 250 µL of 1M Tris-HCl (pH 7.0), 250 µL of 1M Tris-HCl (pH 8.0), 1 mL of 10% w / v SDS, and 200 µL of 0.5M EDTA. Make up to a final volume of 10 mL with deionized water. Store at room temperature for 6 months.

[0116] (10) Polymerase chain reaction (PCR) amplification and fragment screening: Take 20µL of streptavidin M280 magnetic beads (Thermo Fisher, catalog number 11205D) into a 1.5mL centrifuge tube, centrifuge briefly, place the centrifuge tube on a magnetic rack, and remove the supernatant. Then, add 200µL of 1×Binding & Wash buffer, resuspend the magnetic beads and centrifuge briefly again, place the tube on a magnetic rack, remove the supernatant, and wash twice. Then, add 200µL of iBlock buffer, mix well, and incubate at room temperature for 45 minutes. After incubation, centrifuge briefly and remove the supernatant. Then add 200µL of 2×Binding & Wash buffer and wash twice. Next, mix 500ng of ultrasonically sheared genomic DNA with an equal volume of 2×Binding & Wash buffer, incubate with magnetic beads, and incubate at room temperature for 30 minutes to perform a blocking reaction for non-specific DNA fragments. After incubation, add 200µL of 1× Binding & Wash buffer and wash twice. Mix the DNA from step 9 with an equal volume of 2× Binding & Wash buffer and incubate with magnetic beads. Mix thoroughly and incubate with rotation at room temperature for 45 minutes to enrich the biotinylated Bridge Linker adjacent fragments. Subsequently, add 500µL of 0.5% w / v SDS / 2× SSC buffer and rotate at 37°C for 5 minutes, washing five times. Add 500µL of 1× Binding & Wash buffer and rotate at 37°C for 5 minutes, washing twice. After a brief centrifugation, place the tube on a magnetic stand, remove the supernatant, and add 30µL of EB buffer to the magnetic beads. One-third of the beads were used for PCR amplification using 2× High-Fidelity PCR Master Mix (NEB, catalog no. M0541). Primers containing the sample index and primers for Illumina sequencing adapters P5 and P7 were added during amplification using the TruePrep™ Index Kit v2 for Illumina (Vazyme, catalog no. TD202) to facilitate subsequent Illumina sequencing. The PCR amplification program was as follows: heated lid temperature 105°C, initial reaction: 72°C for 3 minutes, 98°C for 30 seconds; cycling: 98°C for 15 seconds, 60°C for 30 seconds, 72°C for 3 minutes; final cycle: 72°C for 5 minutes, and hold at 4°C. The number of PCR cycles depended on the initial sample input; to ensure sequencing quality, no more than 16 cycles were typically performed. One-third of the PCR amplified product was subjected to a 1× cleanup using AMPure XP magnetic beads (Beckman Coulter, catalog no. A63881).After purification, the double-stranded DNA concentration was measured using a Qubit nucleic acid protein quantifier to calculate the total amount of amplified DNA. The remaining 2 / 3 of the beads were used to reference the amount of DNA amplified by PCR using 1 / 3 of the beads. The number of PCR cycles was adjusted to a final total DNA amount of 20 ng for paired-end fragment screening. To meet the Illumina 2×150bp sequencing length requirement, AMPure XP beads were used for 0.8×-0.61× screening, and fragments of 300-600bp were screened for paired-end sequencing. The AMPure XP beads were equilibrated at room temperature for more than 20 minutes. The AMPure XP beads were thoroughly mixed and a 0.61× volume of AMPure XP beads was added to the PCR product. The beads were rotated at room temperature for 10 minutes. After a brief centrifugation, the tube was placed on a magnetic stand and the supernatant was collected in a new tube. Next, add (0.8-0.61) times the volume of Ampure XP magnetic beads to the supernatant. Rotate at room temperature for 10 minutes. After brief centrifugation, place the tube on a magnetic rack and remove the supernatant. Add 800µL of 80% ethanol to wash the beads, remove the supernatant, and repeat the wash. Vacuum dry the beads until cracks form. Finally, immediately add 10µL of EB buffer, mix, and incubate at room temperature with rotation for 10 minutes. Collect the supernatant, which represents the fragment selection product, and measure double-stranded DNA concentration using a Qubit Nucleic Acid Protein Analyzer. To prepare 1× Binding & Wash buffer, add 0.25mL of 1M Tris-HCl pH 7.0, 0.25mL of 1M Tris-HCl pH 8.0, 10mL of 5M NaCl, and 0.05mL of 0.5M EDTA. Make up to a final volume of 50mL with deionized water. Store at room temperature for up to 6 months. Prepare 2× Binding & Wash buffer by adding 0.25 mL of 1M Tris-HCl (pH 7.0), 0.25 mL of 1M Tris-HCl (pH 8.0), 20 mL of 5M NaCl, and 0.1 mL of 0.5M EDTA, and bring the final volume to 50 mL with deionized water. It can be stored at room temperature for up to 6 months. Prepare 0.5% w / v SDS / 2× SSC buffer by adding 5 mL of 2× SSC buffer (Thermo Fisher, catalog number AM9770) and 2.5 mL of 10% w / v SDS, and bring the final volume to 50 mL with deionized water. It can be stored at room temperature for up to 6 months.iBlock buffer is prepared by dissolving 2giBlock protein-based blocking reagent (Thermo Fisher, catalog number T2015) in 90 mL of deionized water. Heat in a 65°C water bath to dissolve. Then, add 5 mL of 10% w / v SDS and bring the final volume to 100 mL with deionized water. It can be stored at room temperature for several months.

[0117] (11) ATAC library construction: After purification, 50 ng of DNA was used for ATAC library construction. PCR amplification reaction was performed using 2× High-Fidelity PCR Master Mix, and primers with sample index and primers for Illumina sequencing adapters P5 and P7 were added during the amplification process using TruePrep™ Index Kit v2 for Illumina for subsequent Illumina sequencing. The PCR amplification program was as follows: hot lid temperature 105°C, initial reaction: 72°C, 3 minutes, 98°C, 30 seconds; cycle reaction: 98°C, 15 seconds; 60°C, 30 seconds; 72°C, 3 minutes; cycle end: 72°C, 5 minutes; 4°C hold, through 14 rounds of PCR amplification reaction to amplify the signal of the target fragment. Subsequently, AMPure XP magnetic beads were used for 1.2×-0.52× purification. The Ampure XP magnetic beads were placed at room temperature for more than 20 minutes to equilibrate. Mix the Ampure XP magnetic beads thoroughly, and add 0.52× volume of Ampure XP magnetic beads to the PCR product, rotate at room temperature for 10 minutes, briefly centrifuge, place the tube on a magnetic stand, and collect the supernatant into a new tube. Then add (1.2-0.52)× volume of Ampure XP magnetic beads to the supernatant, rotate at room temperature for 10 minutes, briefly centrifuge, place the tube on a magnetic stand, and remove the supernatant. Add 800µL 80% ethanol, wash the magnetic beads, remove the supernatant, and repeat the wash. Use a vacuum to dry the magnetic beads until cracks form on the beads. Finally, immediately add 10µL EB buffer, mix well, and rotate and incubate at room temperature for 10 minutes. Collect the supernatant, which is the product after fragment screening. The purified PCR product is used to detect the double-stranded DNA concentration using the Qubit nucleic acid protein quantifier, and the fragment distribution is analyzed using the Qsep-100 analyzer. Figure 4 As shown, the size of DNA fragments in the open chromatin region obtained by Solid ChIA-PET technology ranges from 100 to 3300 bp, mainly concentrated around 900 bp.

[0118] (12) Recover cells (select this step if you need to prepare a single cell suspension, and do not select step (9)): After the Tn5 transposase completes in situ labeling, add Stop buffer (10mM Tris-HCl pH 8.0, 20mM EDTA) and shake at 10 rpm for 5 minutes at room temperature to terminate the transposition reaction. Figure 5 As shown, when the number of cells seeded in the well plate is 200, the cells can still firmly adhere to the surface of the solid object even after multiple rounds of reaction for 3 days. Next, prepare the single cell nucleus suspension. Tilt the well plate to discard the supernatant, add 100µL 0.25% trypsin-EDTA (Thermo Fisher, catalog number 25200056), and incubate at room temperature for 4 minutes. After the incubation, use culture medium containing 10% fetal bovine serum (Excell Bio, catalog number FSP500) to dilute the trypsin, and use a pipette to detach the cells from the solid surface to finally prepare a single cell nucleus suspension ( Figure 6 ).

[0119] (13) Data processing: Run the raw data obtained by sequencing through the pipeline, align it with the genome sequence, and finally obtain interactive data.

[0120] result:

[0121] Table 1 analyzes the ratio of read lengths with linker-tagged sequences to total read lengths in the two experimental strategies of “targeted ligation followed by cleavage” and “targeted cleavage followed by ligation”. The results show that the improved scheme of this application significantly improves the efficiency of obtaining DNA fragments with linker tags.

[0122] Table 1

[0123]

[0124] By using Solid ChIA-PET technology to obtain information on chromatin interactions, target protein enrichment regions, and chromatin open regions: by building a library of 100,000 (0.1 million) cells, such as Figure 7 As shown in the figure, the chromatin interaction loops captured by Solid ChIA-PET technology show significant similarities with the results obtained by traditional ChIA-PET technology (in situ ChIA-PET and Solution ChIA-PET require 1 million and 100 million cells, respectively). At the same time, Solid ChIA-PET technology adopts the strategy of "targeted enzyme cleavage first and then ligation" to specifically act on the chromatin region bound by the target protein. Compared with traditional ChIA-PET technology, this improved solution can more directly capture the real chromatin interactions mediated by specific proteins and effectively reduce non-specific background interference. Figure 7 As shown in Figure 2, the significant enrichment of binding sites on the genome indicates that this technology can specifically capture the chromatin regions mediated by the target protein. In addition, Solid ChIA-PET technology can also effectively obtain relevant information about the open chromatin regions (such as Figure 8 Solid ChIA-PET technology is suitable for analyzing chromatin interactions in a small number of cells, demonstrating its potential for application in small-scale samples.

[0125] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0126] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims, and the specification may be used to interpret the content of the claims.

Claims

1. A sample pre-treatment method, characterized in that: The method comprises the following steps: 1) Cross-linking and fixing the cell or tissue sample, and then performing cell lysis and cell nucleus lysis on the cross-linked and fixed sample; 2) incubating the obtained cell nuclei with a specific antibody for the target protein to prepare a cell nucleus bound by the specific antibody; wherein the target protein is located on chromatin in the cell nucleus; 3) using protein G-micrococcal nuclease to fragment the chromatin in the cell nucleus bound by the heterologous antibody in situ; 4) perform proximity ligation on fragmented chromatin; and 5) Using Tn5 transposase to in situ cut the ligated chromatin and insert a sequencing adapter into the cut site to prepare a cell nucleus containing chromatin with sequencing adapters; Here, before cross-linking and fixation, the method further includes a step of reversibly attaching the cell or tissue sample to a solid surface, or, before incubating the cell nucleus with a specific antibody of the target protein, further includes a step of reversibly attaching the cell nucleus to a solid surface.

2. The method according to claim 1, characterized in that The solid surface is a solid surface treated with polylysine.

3. The method according to claim 1 or 2, characterized in that In step 3), Ca 2+ Activation of the protein G-micrococcal nuclease results in in situ fragmentation of chromatin.

4. The method according to claim 1 or 2, characterized in that In step 4), the fragmented chromatin DNA is blunt-ended with T4 DNA polymerase, the blunt-end repaired DNA ends are A-tailed with Klenow fragment 3'-5' exo-, and the chromatin DNA is adjacently ligated with a biotin-labeled and T-tail-modified oligonucleotide.

5. The method according to claim 1 or 2, characterized in that In step 1), the fixative used for cross-linking fixation includes formaldehyde.

6. The method according to claim 1 or 2, characterized in that In step 1), one or both of the following conditions must be met: (a) lysing the cells by vortexing using a cell lysis buffer, wherein the cell lysis buffer comprises 0.1% w / v SDS; (b) vortexing the resulting solution with a nuclear lysis buffer to lyse the cell nuclei; wherein the nuclear lysis buffer comprises 1% w / v SDS; Optionally, the temperature for the rotation mixing is room temperature, and the time for the rotation mixing is 10 min-25 min.

7. A method for detecting genome-wide chromatin interactions mediated by a target protein, characterized in that: The steps include: Pre-processing the sample using the method according to any one of claims 1 to 6; and The processed samples were used for library construction and high-throughput sequencing.

8. The genome-wide chromatin interaction detection method according to claim 7, characterized in that: Library construction and high-throughput sequencing include the following steps: Reversing cross-linking of the obtained cell nuclei including chromatin with sequencing adapters, purifying DNA, and enriching DNA containing biotin labeling; and The enriched DNA was amplified by PCR, fragment screened, and subjected to high-throughput sequencing.

9. The genome-wide chromatin interaction detection method according to claim 8, characterized in that The cell nuclei, including the chromatin with sequencing adapters, were reversed by using proteinase K at 55°C-65°C.

10. A method for detecting genome-wide chromatin interactions mediated by a target protein in a single cell, characterized in that: The steps include: Pre-processing the sample using the method according to any one of claims 1 to 6; and Single-cell nuclei suspensions were prepared using the processed samples; Optionally, a low-throughput single-cell labeling library is constructed on the single-cell nucleus suspension using a cell sorter, or a high-throughput single-cell labeling library is constructed based on microfluidics technology; Optionally, the cell nuclei including the chromatin with sequencing adapters are separated from the solid surface using trypsin to prepare a single cell nucleus suspension.

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