Chromatin and / or chromosome conformation capture methods and reagents therefor

Abstract

The present invention discloses a method for capturing chromatin and / or chromosome conformation and reagents used therein, and belongs to the field of three-dimensional genomics. The method comprises: subjecting target cells to in vitro cross-linking, lysis, enzymatic digestion with blunt-end restriction endonucleases to add A bases at the ends, and connecting adjacent DNA ends with BP Linker; after labeling the target protein with a specific antibody, using Tn5 fusion protein to target and enrich DNA interactions mediated by the target protein, using biotin labeling to enrich the target fragments of "DNA-tag-DNA" to obtain adjacent ligated DNA fragments, sequencing and analyzing the adjacent ligated DNA fragments, and determining the adjacent ligated DNA bound to the target protein. Compared with traditional methods, the present invention has the advantages of using conventional experimental equipment and a small number of cells, high efficiency, short cycle, low data signal-to-noise ratio, and low cost.

Description

Technical Field

[0001] The present invention relates to the field of three-dimensional genomics, and in particular to a method for capturing chromatin and / or chromosome conformation and reagents used therein. Background Art

[0002] A growing body of research indicates that gene expression is influenced not only by linear regulatory elements but also by spatial interactions within chromatin. Over a decade has passed since the advent of the first chromatin conformation capture (3C) technology, and the ability to detect spatial interactions within chromatin has significantly improved both in terms of capture quality and quantity. Techniques such as circular chromosome conformation capture (4C) and 3C-copy from 3C libraries (5C) have enabled the investigation of long-range interactions within chromatin. The advent of Hi-C (His-C) has been a major breakthrough in the study of the spatial structure of biological genomes. Combined with high-throughput sequencing, it enables genome-wide mapping of chromatin conformation. HiChIP (His-ChIP) and ChIA-PET (ChIA-PET) have taken significant steps toward the detection of spatial interactions mediated by specific proteins. However, currently, efficient, high-resolution, and highly sensitive capture technologies are lacking for the global analysis of three-dimensional chromatin interactions. Techniques such as Hi-C, ChIA-PET, HiChIP, and PLAC-seq not only require large numbers of cells but also suffer from low capture efficiency and cumbersome operation, making them inadequate for sparse cell and tissue samples. Summary of the Invention

[0003] The technical problem to be solved by the present invention is how to capture chromatin and / or chromosome conformation more conveniently and effectively, with a short cycle and / or low data signal-to-noise ratio, and / or how to capture chromatin and / or chromosome conformation in test samples with a small number of cells.

[0004] To solve the above technical problems, the present invention provides a method for capturing chromatin and / or chromosome conformation, named Hi-Tag.

[0005] The method for capturing chromatin and / or chromosome conformation provided by the present invention comprises the following steps:

[0006] S1. Cross-linking target cells in vitro to fix proteins bound to DNA in the target cells to obtain fixed target cells, collecting the fixed target cells and lysing them with a cell lysis buffer to obtain a lysate, and obtaining target cell nuclei from the lysate;

[0007] S2. Enzymatically digesting the DNA in the target cell nucleus with a restriction endonuclease to obtain a target cell nucleus having a blunt-ended DNA, and adding an A base to the 3' end of the blunt-ended DNA to obtain a target cell nucleus having an A base-terminated DNA;

[0008] S3. Use T4 DNA ligase to connect the DNA with A base end and BP Linker to obtain the target cell nucleus containing adjacent ligated DNA;

[0009] The BP Linker is a double-stranded DNA with a sticky end having a protruding nucleotide T at the 3' end. The nucleotide sequence of one chain of the BP Linker is 5'-CGCGATATCTTATCTGACT-3' (sequence 1), and the nucleotide sequence of the other chain is 5'-GTCAGATAAGATATCGCGT-3' (sequence 2). The deoxythymidylate T at position 10 of sequence 1 is modified with biotin. The 3', 5'-phosphodiester bond between the two nucleotides of each chain of the two chains of the BP Linker ( Figure 2 (indicated by the arrow in the lower left figure) are replaced by 3', 5'-phosphothioate diester bonds ( Figure 2 As shown by the arrow in the right figure in the lower middle figure, the sulfur atom replaces one of the oxygen atoms in the phosphodiester bond);

[0010] S4, capturing the target cell nucleus containing the proximity-linked DNA, reacting a specific antibody against the target protein with the target cell nucleus containing the proximity-linked DNA to obtain a target cell nucleus bound to a primary antibody, binding the target cell nucleus bound to the primary antibody with a secondary antibody against the specific antibody to obtain a target cell nucleus bound to the primary antibody and the secondary antibody, reacting the target cell nucleus bound to the primary antibody and the secondary antibody with a Tn5 fusion protein to obtain an in situ tagged target cell nucleus, wherein the in situ tagged target cell nucleus is a cell nucleus bound to the Tn5 fusion protein, activating the Tn5 fusion protein in the in situ tagged target cell nucleus with magnesium ions, causing the proximity-linked DNA bound to the target protein to be fragmented to obtain a target cell nucleus containing proximity-linked DNA fragments, extracting and purifying DNA from the target cell nucleus containing the proximity-linked DNA fragments to obtain a mixed DNA containing proximity-linked DNA fragments, capturing the proximity-linked DNA fragments from the mixed DNA to obtain the proximity-linked DNA fragments;

[0011] S5. Perform sequencing analysis on the proximity-linked DNA fragments to determine the proximity-linked DNA bound to the target protein.

[0012] In the above method, in S3, the DNA with an A base terminus can be ligated with BP Linker using T4 DNA ligase in a ligation system. In the ligation system, the BP-Linker content is 0.8 ng / μl and the T4 DNA ligase content is 2 U / μl. In S3, the ligation system can be composed of 0.8 ng / μl BP-Linker, 2 U / μl T4 DNA ligase, 0.1 mg / ml BSA, 10 g / L Trition X-100, and 1× T4 DNA ligase Buffer. In S3, the ligation of the DNA with an A base terminus with BP Linker using T4 DNA ligase can be allowed to react at 25°C for 4 hours.

[0013] In S1, the in vitro cross-linking is performed using formaldehyde. For example, the target cells can be treated in a 1% formaldehyde solution for 10 minutes. The solvent of the formaldehyde solution is PBS, and the solute is formaldehyde. The composition of the PBS is 1mM KH2PO4, 155mM NaCl, 3mM Na2HPO4-7H2O, and the rest is water; the pH of the PBS is 7.4. S1 includes performing cross-linking using formaldehyde and then terminating the cross-linking with a 0.125M glycine solution (the solvent is water). The number of cells can be 1×10 4 -1×10 6 can be freely adjusted according to the experimental samples.

[0014] In S1, the cell lysate is composed of a solute and a solvent, the solvent is water, and the solute is composed of Tris-HCl, NaCl, polyoxyethylene octylphenol ether, and a protease inhibitor. In the cell lysate, the content of Tris-HCl is 10mM, the content of NaCl can be 10mM, the content of polyoxyethylene octylphenol ether can be 2mL / L, and the content of protease inhibitors can be 10g / L. In S1, obtaining the target cell nucleus from the lysate can include adding the lysate to a buffer solution containing SDS, incubating the loosened chromatin at 62°C, and then adding Triton X-100 to neutralize the SDS, wherein the content of SDS in the buffer solution containing SDS is 3-5g / L.

[0015] In S1, the SDS-containing buffer solution may be composed of 1× rCutsmart buffer, 3-5 g / L SDS, and water. The amount of Triton X-100 added may satisfy the content of Triton X-100 in the reaction system of 1.67 g / L.

[0016] In S1, the time for loosening chromatin is 10 minutes. The neutralization can be performed by incubating at 20-25° C. for 5 minutes and then incubating on ice for 10 minutes.

[0017] The above method does not include the step of ultrasonication.

[0018] In S2, the restriction endonuclease is a restriction endonuclease that produces blunt ends, such as any one of HeaIII and AluI. The enzymatic digestion can be carried out at 37°C for 6-8 hours. The enzymatic digestion can be carried out in a reaction system with a restriction endonuclease content of 1.0 U / μl. The reaction system consists of a solute and a solvent, wherein the solute is 1× rCutSmart buffer, 10 g / L Trition X-100, and 1.0 U / μl of the restriction endonuclease, and the solvent is water.

[0019] The addition of an A base to the 3' end of the blunt-ended DNA is performed using Klenow Exo-5'-3' enzyme. Specifically, the addition of an A base to the 3' end of the blunt-ended DNA can be performed by incubating the reaction system at 37°C for 1 hour. The A addition reaction system can be composed of: 1×NEB Buffer 2, 0.2mM dATP, 10g / L Trition X-100, 1.0U / μl Klenow Exo-5'→3' (NEB, M0212L), and water.

[0020] In S3, T4 DNA ligase is used to connect the DNA having an A base end and BP Linker in a ligation system. In the ligation system, the content of the BP-Linker is 0.8 ng / μl, and the content of the T4 DNA ligase is 2 U / μl.

[0021] In S3, the ligation system may be composed of 0.8 ng / μl of the BP-Linker, 2 U / μl of T4 DNA ligase, 0.1 mg / ml of BSA, 10 g / L of Trition X-100, and 1×T4 DNA ligase buffer.

[0022] In S3, the DNA having an A base end and the BP Linker are connected using T4 DNA ligase at 25° C. for 4 hours or at 16° C. for 8-10 hours.

[0023] In S4, the Tn5 fusion protein may be ProteinG-Tn5, ProteinA-Tn5 or proteinAG-Tn5, wherein the ProteinG-Tn5 is a fusion protein composed of proteinG and Tn5 transposase; the ProteinA-Tn5 is a fusion protein composed of proteinA and Tn5 transposase; and the ProteinAG-Tn5 is a fusion protein composed of proteinA, proteinG and Tn5 transposase.

[0024] In said S4, before extracting and purifying DNA from the target cell nucleus containing the adjacently linked DNA fragments, the step of terminating the cleavage reaction of the Tn5 fusion protein with proteinase K and SDS and digesting the protein is included.

[0025] In S4, capturing the proximity-ligated DNA fragments from the mixed DNA comprises enriching the proximity-ligated DNA fragments using a streptavidin-modified solid.

[0026] The streptavidin-modified solid may be streptavidin magnetic beads (streptavidin-modified magnetic beads).

[0027] The present invention also provides a reagent for capturing chromatin and / or chromosome conformation, the reagent comprising any one of the above-mentioned BP Linkers and at least one of the following substances: the above-mentioned Tn5 fusion protein, formaldehyde, cell lysate, a restriction endonuclease that produces blunt ends, Klenow Exo - 5'→3' and streptavidin-modified solids.

[0028] The present invention also protects the above-mentioned BP Linker.

[0029] The present invention also protects the use of the above-mentioned BP Linker in chromatin and / or chromosome conformation capture, or the use of a reagent for chromatin and / or chromosome conformation capture in chromatin and / or chromosome conformation capture.

[0030] like Figure 1 As shown, the Hi-Tag chromatin and / or chromosome conformation capture method of the present invention first uses a blunt-end restriction endonuclease to cut chromatin, then adds an A base to the cut DNA end. Then, the present invention innovatively uses a bridge DNA linker (BP Linker) with both biotin and thiolation modifications to connect adjacent DNA ends under the action of T4 DNA ligase. After labeling the target protein with a specific antibody, the Tn5 fusion protein is innovatively used to target and enrich the target protein-mediated DNA interaction. Subsequently, biotin labeling is used to enrich the "DNA-tag-DNA" target fragment. Finally, a single-step library amplification is required to generate a sequencing library.

[0031] The advantages of the present invention over traditional HiChIP, ChIA-PET and PLAC-seq methods are as follows: the present invention does not require any complex instruments such as ultrasonic disruptors, and all operations can be completed with conventional laboratory equipment; the present invention can be performed on 10,000-1,000,000 cells, which has significant advantages for experimental samples with a small number of cells; the present invention can efficiently capture DNA spatial interactions mediated by specific proteins, and sequencing samples can be directly obtained through Tn5 fusion protein, which greatly shortens the experimental cycle and can obtain samples to be sequenced in only 1.5 days; the present invention can obtain a chromatin spatial structure map equivalent to that of traditional technologies with a relatively small amount of data, greatly saving sequencing costs; the data output by the present invention has a higher signal-to-noise ratio than traditional methods, and the use of a BP linker doubly modified with biotin and thiolate ensures that Tn5 will not cut the linker during in situ cutting, thereby greatly improving the proportion of valid data in the data. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Flowchart for constructing Hi-Tag sequencing library.

[0033] Figure 2 Figure 2 shows a bridge DNA linker with biotin and thiolation modifications. Arrows indicate the substituted atoms.

[0034] Figure 3 The Agilent 2100 instrument detects the distribution of DNA fragments after genomic fragmentation by restriction endonuclease AluI.

[0035] Figure 4 The cell nuclei are adsorbed on concanavalin-coated magnetic beads.

[0036] Figure 5 Correlation analysis between Hi-Tag method and HiC and HiChIP data.

[0037] Figure 6 For visualization, the chromatin interaction map of the GATA1 gene location identified by HiChIP and Hi-Tag technologies.

[0038] Figure 7 The enrichment of CUT&Tag signals within the loop anchor and random regions (5kb) identified by Hi-Tag technology (line graph on the left); the overlap of peaks identified by Hi-Tag technology and CUT&Tag (bar graph on the right).

[0039] Figure 8 The reliability (Macs2 score) of the overlapping and non-overlapping parts of the peaks identified by Hi-Tag technology and CUT&Tag results.

[0040] Figure 9 The loop anchors covering histone binding sites identified using the CUT&Tag peak as a reference and the Hi-Tag 1D peak as a reference.

[0041] Figure 10 The distribution of chromatin interaction distances when the CUT&Tag peak is used as a reference and the 1D peak of Hi-Tag is used as a reference.

[0042] Figure 11 For visualization, the loops associated with GATA1 were identified using the CUT&Tag peak as a reference and the Hi-Tag 1D peak as a reference. DETAILED DESCRIPTION

[0043] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0044] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0045] Example 1: Hi-Tag Chromatin and / or Chromosome Conformation Capture Method

[0046] The following uses 500,000 K562 cells and histone H3K27ac as a target protein as an example to illustrate the method of capturing Hi-Tag chromatin and / or chromosome conformation of the present invention. The method comprises the following steps:

[0047] S1. Crosslink the target cells in vitro to fix the DNA-bound proteins in the target cells, obtaining fixed target cells. Collect the fixed target cells and lyse them with a lysis buffer to obtain a lysate, from which the target cell nuclei are obtained. This step takes 1 hour. The details are as follows:

[0048] S11. Collect the cultured K562 cells by centrifugation and resuspend 500,000 K562 cells in 30 ml of a 1% formaldehyde solution (composed of a solute and a solvent, with the solute being formaldehyde and the solvent being PBS) and incubate at 25°C for 5 minutes. Immediately thereafter, add an aqueous glycine solution to a glycine concentration of 0.125 M in the reaction system. Incubate at 25°C for 10 minutes to terminate the cross-linking reaction. The fixed cells were collected by centrifugation at 2500 g for 5 minutes at 4°C and then rinsed once with 4°C pre-chilled PBS to obtain the fixed cells. The fixed cells can be used for experiments immediately or stored at -80°C. The composition of the PBS is 1 mM KH2PO4, 155 mM NaCl, 3 mM Na2HPO4·7H2O, and the remainder is water; the pH of the PBS is 7.4.

[0049] S12. Add ice-cold cell lysis buffer to the fixed target cells, react on ice for 15 minutes to lyse the cells to obtain a lysate, and obtain the target cell nucleus from the lysate according to the following steps: resuspend the lysate in 1 ml of SDS-containing buffer solution (composed of 5 g / L SDS, 1×rcutSmart buffer (NEB, product number B6004S) and water) and incubate at 62°C for 10 minutes to loosen the chromatin, then immediately add 200 μL of 10% TritionX-100 aqueous solution and incubate at 25°C for 5 minutes and then transfer to ice and incubate for 10 minutes to neutralize the SDS in the system. Collect the target cell nuclei by centrifugation, wash them once with 1×rCutSmart buffer and PBST (PBST is a buffer solution composed of solute and solvent, the solvent is the above-mentioned PBS, the solute is Tween-20, and the content of Tween-20 in PBST is 1 g / L) to obtain the target cell nucleus.

[0050] Wherein, the pH of cell lysate is 8.0, is a solution consisting of solute and solvent, the solvent is water, and the solute consists of Tris-HCl, NaCl, polyoxyethylene octyl alkyl phenol ether and protease inhibitors. In the cell lysate, the content of Tris-HCl is 10mM, the content of NaCl is 10mM, the content of polyoxyethylene octyl alkyl phenol ether is 2mL / L, and the content of protease inhibitors is 10g / L. Wherein, polyoxyethylene octyl alkyl phenol ether is Igepal CA630 (Beyotime company product number is ST2045-500ml, CAS number is 9002-93-1). Protease inhibitors are products of Sigma company, and product number is 11873580001.

[0051] S2. Using a restriction endonuclease, the DNA in the target cell nucleus is digested to produce blunt-ended DNA in the target cell nucleus. A base is added to the 3' end of the blunt-ended DNA to obtain A-terminated DNA in the target cell nucleus. This step takes 5 hours. The details are as follows:

[0052] S21. Resuspend the target cell nucleus in 1 ml of enzyme digestion system and incubate at 37°C for 4 hours. You can extract DNA from a small amount of sample to detect whether the restriction endonuclease has sufficiently cut the genome. Taking AluI as an example, the genome fragment after cutting should be around 2kb. The details are as follows: Resuspend the target cell nucleus in 1 ml of enzyme digestion system (the enzyme digestion system consists of 1× rCutSamrt buffer, 10g / L Trition X-100 and 1.0U / μl AluI), incubate at 37°C for 4 hours, and the genome fragment after cutting should be around 2kb (such as Figure 3 shown).

[0053] S22. Collect the target cell nuclei by centrifugation and wash once with 1× NEB Buffer 2 (NEB, B0212L). Resuspend the target cell nuclei in 250 μl of the A-addition reaction system and incubate with rotation at 37°C for 1 hour to obtain target cell nuclei with A-terminated DNA. The 250 μl A-addition reaction system consists of: 1× NEB Buffer 2, 0.2 mM dATP, 10 g / L TritionX-100, and 1.0 U / μl Klenow Exo-5'→3' (NEB, M0212L).

[0054] S3. Use T4 DNA ligase to ligate the A-terminated DNA to the BP Linker to obtain the target cell nucleus containing the adjacently linked DNA. This step takes 4 hours. The details are as follows:

[0055] The target cell nuclei were collected by centrifugation and washed once with PBST and once with 1× T4 DNA ligase buffer (NEB, B0202L). The target cell nuclei were resuspended in 1 ml of ligation system and incubated with rotation at 16°C for 4 hours to obtain the target cell nuclei containing adjacent ligated DNA.

[0056] The ligation system consisted of 0.8 ng / μl BP-Linker, 2 U / μl T4 DNA ligase (NEB, M0202L), 0.1 mg / ml BSA, 10 g / L Trition X-100 and 1×T4 DNA ligase buffer.

[0057] Among them, the structure of BP-Linker is as follows Figure 2As shown, BP Linker is a double-stranded DNA with a sticky end with a protruding nucleotide T at the 3' end. The nucleotide sequence of one strand of BP Linker is 5'

[0058] -CGCGATATCTTATCTGACT-3' (sequence 1), the nucleotide sequence of the other chain is 5'

[0059] -GTCAGATAAGATATCGCGT-3' (sequence 2), the deoxythymidine T (dT) at position 10 of sequence 1 is modified with biotin. The 3', 5'-phosphodiester bond between the two nucleotides of each chain of the two chains of BP Linker ( Figure 2 (indicated by the arrow in the lower left figure) are replaced by 3', 5'-phosphothioate diester bonds ( Figure 2 As shown by the arrow in the lower right figure, the sulfur atom replaces one of the oxygen atoms in the phosphodiester bond).

[0060] S4, capture the target cell nucleus containing the adjacent connected DNA, react the specific antibody against the target protein with the target cell nucleus containing the adjacent connected DNA, obtain the target cell nucleus in combination with an antibody, combine the target cell nucleus in combination with the primary antibody with the secondary antibody against the specific antibody, obtain the target cell nucleus in combination with the primary and secondary antibodies, react the target cell nucleus in combination with the primary and secondary antibodies with the Tn5 fusion protein, obtain in situ labeled target cell nucleus, the in situ labeled target cell nucleus is the cell nucleus in combination with the Tn5 fusion protein, activate the Tn5 fusion protein in the in situ labeled target cell nucleus with magnesium ions, fragment the adjacent connected DNA bound to the target protein, obtain the target cell nucleus containing the adjacent connected DNA fragments, extract and purify DNA from the target cell nucleus containing the adjacent connected DNA fragments, obtain a mixed DNA containing the adjacent connected DNA fragments, capture the adjacent connected DNA fragments from the mixed DNA, and obtain the adjacent connected DNA fragments. The time required for this step is 7 hours. Specific as follows:

[0061] S41. Capture the target cell nucleus containing the adjacent ligated DNA. The specific operation is as follows: collect the target cell nucleus by centrifugation, wash it once with washing buffer, then resuspend it with 500 μl washing buffer and transfer it to a new 1.5 ml low-absorption EP tube, add 50 μl ConA beads solution and gently pipette to mix, incubate at 25°C for 10 minutes to allow ConA beads to fully bind to the cell nucleus (in this step, some samples can be taken out and placed under a microscope for observation. Under normal circumstances, more than 98% of the cell nuclei will be captured by beads. Figure 4 ), capturing target cell nuclei containing adjacently linked DNA.

[0062] The washing buffer consisted of pH 7.5, 0.02 M HEPES buffer, 0.15 M NaCl, 0.5 mM Spermidine, and 1× protease inhibitor (Sigma, 11873580001).

[0063] ConA bead solution was prepared as follows: 50 μl of ConA beads (BioMap, BP531) (concanavalin A (ConA)-coated magnetic beads) were equilibrated at 25°C for 30 minutes. After mixing, 50 μl was removed and added to 500 μl of binding buffer, pipetting and mixing thoroughly. The beads were then placed on a magnetic stand and allowed to stand for 2 minutes. The supernatant was removed and the solution was repeated once. The beads were then resuspended in 50 μl of binding buffer to prepare the ConA bead solution. The binding buffer was a buffer solution composed of HEPES (pH 7.5, 0.02 M), KCl (0.01 M), NaCl (0.01 M), and MnCl2 (0.01 M).

[0064] S42. React the specific antibody against the target protein with the target cell nucleus containing adjacently linked DNA to obtain the target cell nucleus bound to the primary antibody. The specific operation is as follows: Place the EP tube on a magnetic stand and let it stand for 2 minutes. Remove the supernatant and resuspend it in 500 μl of antibody buffer premixed with the specific antibody (the dilution ratio of the antibody can refer to the immunofluorescence dilution ratio in the antibody manual; the dilution ratio in this case is 1:100). Incubate with rotation at 25°C for 1 hour to obtain the target cell nucleus bound to the primary antibody.

[0065] The composition of the antibody buffer premixed with specific antibodies is as follows: 20 mM HEPES pH 7.5, 150 mM NaCl, 12.5 μL 0.5 mM spermidine, 0.05% Digitonin, 2 mM EDTA, 0.1% BSA and 2 μg / 100 μL anti-H3K27ac (abcam, ab4729).

[0066] S43. Allow the target cell nuclei bound to the primary antibody to bind to the secondary antibody against the specific antibody, thereby obtaining the target cell nuclei bound to the primary and secondary antibodies. Specifically, place the EP tube on a magnetic stand for 2 minutes. Remove the supernatant and resuspend in 500 μl of Dig-wash buffer premixed with the secondary antibody. Incubate at 25°C with rotation for 1 hour to obtain the target cell nuclei bound to the primary and secondary antibodies.

[0067] The composition of Dig-wash buffer is: pH 7.5, 0.02M HEPES buffer, 0.15M NaCl

[0068] , 0.5 mM Spermidine, 0.5 g / L Digitonin, and 1× protease inhibitor (Sigma, 11873580001). Dig-wash buffer premixed with secondary antibody consists of goat anti-rabbit IgG H&L (abcam, ab6702) and Dig-wash buffer. The content of goat anti-rabbit IgG H&L in the Dig-wash buffer premixed with secondary antibody is 0.02 μg / μL.

[0069] S44. React the target cell nuclei bound to the primary and secondary antibodies with the Tn5 fusion protein to obtain in situ labeled target cell nuclei. The specific steps are as follows: Place the EP tube on a magnetic stand and let it stand for 2 minutes. Remove the supernatant and wash three times with 1 ml of Dig-wash buffer. Resuspend in 500 μl of Dig-300 buffer premixed with a final concentration of 0.8 μM ProteinG / A-Tn5 fusion protein (Novagen TD901). Incubate with rotation at room temperature for 1 hour to obtain in situ labeled target cell nuclei.

[0070] The composition of Dig-300 buffer is: pH 7.5, 0.02M HEPES buffer, 0.3M NaCl, 0.5mM Spermidine, 0.1g / L Digitonin, 0.8μM ProteinG / A-Tn5 fusion protein and 1× protease inhibitor (Sigma, 11873580001).

[0071] S45. Use magnesium ions to activate the Tn5 fusion protein in the target cell nuclei for in situ tagmentation, fragmenting the proximity-ligated DNA bound to the target protein. This yields target cell nuclei containing proximity-ligated DNA fragments. The procedure is as follows: Place the EP tube on a magnetic stand for 2 minutes. Remove the supernatant and wash three times with 1 ml of Dig-300 buffer. Resuspend in 500 μl of Tagmentation buffer and incubate with rotation at 37°C for 1 hour to yield target cell nuclei containing proximity-ligated DNA fragments.

[0072] The composition of the tagmentation buffer is 20 mM HEPES pH 7.5, 300 mM NaCl, 12.5 μM spermidine, 0.01% Digitonin and 0.1 mM MgCl2.

[0073] S46. Extract and purify DNA from the target cell nuclei containing adjacently linked DNA fragments to obtain a mixed DNA containing adjacently linked DNA fragments. The specific procedure is as follows: Add 10 μl of 100 g / L SDS solution and 10 μl of proteinase K solution and incubate at 55°C for 30 minutes to terminate the transposase reaction and digest the protein. After the reaction, add an equal volume of phenol-chloroform-isoamyl solution (phenol:chloroform:isoamyl alcohol = 25:24:1). Mix vigorously by inversion, then centrifuge at 15,000 rpm for 5 minutes at room temperature. Transfer the supernatant to a new 1.5 mL EP tube. Add 50 μl of 3 M sodium acetate solution, 2 μl of 10 mg / ml glycogen solution, and 550 μl of pre-chilled isopropanol solution in that order. Mix by inversion, and then let it settle at -20°C for 1 hour. Centrifuge at 15,000 rpm and 4°C for 30 minutes, remove the supernatant, wash the precipitate with 1 ml of 75% ethanol (freshly prepared) solution, centrifuge at 15,000 rpm and 4°C for 5 minutes, remove as much supernatant as possible, place the precipitate on a clean bench to dry until transparent, add 200 μl of nuclease-free water to dissolve the precipitate, and place on ice until used to obtain a mixed DNA solution containing adjacent-linked DNA fragments.

[0074] S47. Capture the proximity-ligated DNA fragments from the mixed DNA to obtain the proximity-ligated DNA fragments. Specifically, place 30 μl of M-280 Streptavidin magnetic beads (ThermoFisher, 11205D) on a magnetic stand and let stand for 2 minutes. Discard the liquid and add 200 μl of Binding Buffer (composed of 10 mM Tris-HCl pH 7.5, 1 mM EDTA, and 2 M NaCl) to resuspend the beads. Incubate with rotation at 25°C for 5 minutes. After discarding the Binding Buffer, wash the streptavidin beads twice with 200 μl of Wash Buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, and 1 M NaCl). Mix the DNA solution containing the proximity-ligated DNA fragments from S46 with an equal volume of Binding Buffer and resuspend the washed streptavidin beads. Incubate with rotation at 25°C for 30 minutes to enrich the biotinylated target fragments (proximity-ligated DNA fragments). Place the reaction system on a magnetic stand for 2 minutes, then discard all liquid. Wash the beads three times with 500 μl of 0.5% SDS / 2×SSC buffer (ThermoFisher, 15557044) and once with 200 μl of Wash Buffer. Resuspend the washed beads in 30 μl of nuclease-free water and place on ice until ready to use. This yields magnetic beads immobilized with proximity-ligated DNA fragments.

[0075] S5. Sequencing and analyzing the proximity-linked DNA fragments to determine the proximity-linked DNA that binds to the target protein. The specific method is as follows:

[0076] S51. Take 10 μl of the magnetic beads immobilized with the adjacent ligated DNA fragments in S47 and add them to the amplification system. The number of PCR cycles is controlled within 15.

[0077] The primer sequences are as follows:

[0078] P5 PCR Primer for Illumina:

[0079] 5'-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTC-3',

[0080] P7 PCR Primer for Illumina:

[0081] 5'-CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGGGGCTCGG-3', N is A, T, C or G; 8 Ns are indexes.

[0082] 1) Equilibrate AMPure XP beads to room temperature. After amplification, transfer the PCR mixture to a 1.5ml low-binding EP tube and add 1.5 times the volume of AMPure XP beads. Vortex to mix thoroughly and incubate at room temperature for 10 minutes. Place the EP tube on a magnetic rack for 5 minutes. Once the liquid has clarified, remove all liquid. While the EP tube remains on the magnetic rack, add 500μl of freshly prepared 80% ethanol solution and incubate for 1 minute before removing (repeat this process once). After removing all liquid, allow the beads to air dry. Once the beads are non-reflective, add 30μl of nuclease-free solution to elute the DNA. Determine the concentration of the eluted DNA solution using a Qubit 3.0 Fluorometer. Determine the number of amplification cycles for the remaining 20μl of beads based on sequencing requirements.

[0083] 2) In accordance with the requirements of next-generation sequencing, the final sequencing library was screened for fragments ranging from 200 to 1000 bp using AMPure XP beads. First, add 0.6 times the volume of AMPure XP beads to the DNA solution, vortex to mix, and incubate at room temperature for 10 minutes. The reaction system was then placed on a magnetic stand and allowed to stand for 5 minutes. After the liquid clarified, the supernatant was transferred to a new 1.5mL EP tube and 0.15 times the initial volume of AMPure XP beads was added. Vortex to mix, and incubate at room temperature for 10 minutes. Subsequent washing and DNA elution steps were consistent with those in S47. Before sequencing, the library was estimated to have a valid fragment ratio. A small amount of the library was added to the restriction endonuclease EcoRV. Due to the presence of this restriction site in the linker sequence, the DNA connected by the linker was cut into two ends, and the fragment distribution would be significantly changed. The raw sequencing data was obtained.

[0084] S52, Hi-Tag data analysis

[0085] 1Hi-Tag data analysis process

[0086] 1.1Adapter and linker filtering

[0087] The raw sequencing data obtained by S51 were quality controlled to remove low-quality sequencing reads. Subsequently, for each sample, the barcode corresponding to the cell was obtained using the P5, P7, and 10X barcode sequences. After that, the adapter was removed using trim_galore. The specific command is trim_galore-q 20--phred33--paired--Nextera--trim-n--gzip${reads[0]}${reads[1]}. The raw sequence and linker were then aligned using the trimLinker command (ChIA-PET2). The specific command is trimLinker-e 2-t 12-m 1-k 1-l16-o$RESULTS-n$SAMPLE-A ACGCGATATCTTATC-B AGTCAGATAAGATAT. The sequences on both sides of the linker were extracted, which are referred to as PETs (Pair-End-Tags) below. If at least one of the double-ended sequences contains a linker, the linker is removed and the sequence from the 5' end to the start of the linker is retained. Non-chimeric PETs (DNA sequences at both ends of the screened linker) are used for the next step of analysis.

[0088] 1.2 Sequence alignment

[0089] Use BWA software to align the PETs obtained in the previous step to the GRCh38 reference genome. The parameters used are bwa mem-SP5M. The specific command is bwa mem-SP5M -t 20${params.bwa_index_prefix}${reads[0]}${reads[1]}>${sample_id}_mem.sam, and further obtain the resulting bam file after alignment.

[0090] 1.3 Data Post-Processing

[0091] First, the PETs alignment results obtained in the previous step are parsed into interaction pairs, hereinafter referred to as interaction pairs (pairs). Due to the PCR amplification step in the experiment, there is information redundancy in the pairs, which needs to be removed during the analysis process. Subsequently, in order to filter out the noise in the data (multiple alignments, single-end alignments, etc.), the bam file obtained in the previous step is parsed and classified in combination with the enzyme cutting site information. Interaction sequences with valid and unique alignments ("unique-unique (UU)" pairs) and MAPQ values greater than 30 are retained, and a .pairs file that conforms to the 4D Nucleome (4DN) Alliance is output for subsequent analysis. The above steps are completed using the pairtools tool, and the specific parsing command is pairtools parse-c$GENOME_SZ-o$RESULTS / ${SAMPLE}.pairs.gz

[0092] --drop-sam $RESULTS / ${SAMPLE}.sam The command to select UU pairs is pairtoolsselect '(pair_type == "UU")' -o $RESULTS / ${SAMPLE}.sorted.dedup.UU.pairs.gz $RESULTS / ${SAMPLE}.sorted.dedup.pairs.gz . The output interaction pair .pairs file is in a standard format and contains readID information, interaction position information, plus and minus strand information, etc.

[0093] 1.4 Identification of significant interactions

[0094] The distribution of pairs obtained in the previous step may be affected by the distribution of restriction sites and target protein binding regions, leading to biased identification of interaction regions. FitHiChIP software was used to correct for this and identify significant interactions using coverage bias regression of FitHiChIP(L) and FitHiChIP(L+M). The command was bashFitHiChIP_HiCPro.sh -C configfile_BiasCorrection_CoverageBias.txt. The significance coefficient was set to the default value (FDRThr: FDR threshold for FitHiChIP loop significance (default = 0.01)). The software identified loops with at least one end enriched for interaction with the CUT&Tag (ChIP-seq) peak, making the resulting loops more accurate and reliable, reducing false positives.

[0095] 2 Interactive capture of K562 cell line using Hi-Tag technology

[0096] 2.1 K562 cell line Hi-Tag data statistics

[0097] Analysis of Hi-Tag data at different scales, obtained by random downsampling, revealed that valid reads containing linkers accounted for 82% of the total raw data. Uniquely aligned reads accounted for approximately 30% of the raw data, with cis interactions accounting for approximately 74% and trans interactions accounting for approximately 25% (Table 1). The number of identified loops increased with the amount of raw data.

[0098] Table 1. K562 cell line Hi-Tag data statistics

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[0100]

[0101] 3. Comparison with existing HiChIP technology

[0102] In the K562 cell line, even with lower cell counts and sequencing depth, Hi-Tag can obtain chromatin interaction maps that are very similar to HiC and HiChIP data (e.g. Figure 5 One example is a loop associated with the marker gene GATA1 of the K562 cell line (as shown in Figure 6 shown).

[0103] By comparing the CUT&Tag enrichment signals inside the loop anchor and the random 5kb region, it was found that the signal inside the loop anchor was much higher than that in the random region (such as Figure 7 Moreover, the peaks identified by Hi-Tag overlapped with the peaks of CUT&Tag by nearly 90%, and the credibility of the overlapping peaks was much higher than that of the non-overlapping peaks (as shown in Figure 2). Figure 8 and 9 Therefore, using Hi-Tag data can obtain genome-wide protein binding sites similar to CUT&Tag or ChIP-seq.

[0104] In addition, without considering the interaction, Hi-Tag data can be regarded as one-dimensional data similar to ChIP-seq, and peaks can be identified based on this. By comparison, it was found that Hi-Tag technology can be stable in data of different orders of magnitude. When the peaks identified by its own data are used as a reference, more accurate interaction pairs can be obtained. In addition, the characteristics of loop anchors are similar to the results of using CUT&Tag peaks as a reference. More than 90% of loop anchors have peaks at both ends (such as Figure 10 As shown in ), the distance distribution of loops is also very similar (as shown in Figure 10 Similar interaction patterns can also be found using visualization tools in the GATA1 example (as shown in Figure 11 In summary, Hi-Tag can simultaneously generate a genome-wide map of specific protein binding sites and a map of chromatin interactions.

[0105] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A method for capturing chromatin and / or chromosome conformation, characterized in that: The method comprises the following steps: S1. Cross-linking target cells in vitro to fix proteins bound to DNA in the target cells to obtain fixed target cells, collecting the fixed target cells and lysing them with a cell lysis buffer to obtain a lysate, and obtaining target cell nuclei from the lysate; obtaining the target cell nuclei from the lysate comprises adding the lysate to a buffer solution containing SDS to incubate at 62° C. to loosen chromatin, and then adding Triton X-100 to neutralize the SDS, wherein the SDS content in the buffer solution containing SDS is 3-5 g / L; S2. Enzymatically digesting the DNA in the target cell nucleus with a restriction endonuclease to obtain a target cell nucleus having a blunt-ended DNA, and adding an A base to the 3' end of the blunt-ended DNA to obtain a target cell nucleus having an A base-terminated DNA; S3. Use T4 DNA ligase to connect the DNA with A base end and BP Linker to obtain the target cell nucleus containing adjacent ligated DNA; The BP Linker is a double-stranded DNA having a sticky end with a protruding nucleotide T at the 3' end. The nucleotide sequence of one chain of the BP Linker is Sequence 1 in the sequence listing, and the nucleotide sequence of the other chain is Sequence 2 in the sequence listing. The deoxythymidylate T at position 10 of Sequence 1 is modified with biotin, and the 3', 5'-phosphodiester bond between the two nucleotides in each chain of the two chains of the BP Linker is replaced with a 3', 5'-phosphothioate diester bond. S4, capturing the target cell nucleus containing the proximity-linked DNA, reacting a specific antibody against the target protein with the target cell nucleus containing the proximity-linked DNA to obtain a target cell nucleus bound to a primary antibody, binding the target cell nucleus bound to the primary antibody with a secondary antibody against the specific antibody to obtain a target cell nucleus bound to the primary antibody and the secondary antibody, reacting the target cell nucleus bound to the primary antibody and the secondary antibody with a Tn5 fusion protein to obtain an in situ tagged target cell nucleus, wherein the in situ tagged target cell nucleus is a cell nucleus bound to the Tn5 fusion protein, activating the Tn5 fusion protein in the in situ tagged target cell nucleus with magnesium ions, causing the proximity-linked DNA bound to the target protein to be fragmented to obtain a target cell nucleus containing proximity-linked DNA fragments, extracting and purifying DNA from the target cell nucleus containing the proximity-linked DNA fragments to obtain a mixed DNA containing proximity-linked DNA fragments, capturing the proximity-linked DNA fragments from the mixed DNA to obtain the proximity-linked DNA fragments; S5. Perform sequencing analysis on the proximity-linked DNA fragments to determine the proximity-linked DNA bound to the target protein.

2. The method according to claim 1, wherein: In S3, the DNA having an A base end is connected to the BP Linker using T4 DNA ligase in a ligation system. In the ligation system, the content of the BP-Linker is 0.8 ng / μl, and the content of the T4 DNA ligase is 2 U / μl.

3. The method according to claim 1 or 2, characterized in that: In S1, the in vitro cross-linking is performed using formaldehyde.

4. The method according to claim 1 or 2, characterized in that: In said S4, before extracting and purifying DNA from the target cell nucleus containing the adjacently linked DNA fragments, the step of terminating the cleavage reaction of the Tn5 fusion protein with proteinase K and SDS and digesting the protein is included.

5. The method according to claim 1 or 2, characterized in that: In S4, capturing the proximity-ligated DNA fragments from the mixed DNA includes enriching the proximity-ligated DNA fragments using a streptavidin-modified solid.

6. The method according to claim 1 or 2, characterized in that The method does not include a sonication step.

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