Chromatin conformation capturing method of frozen tissue

Abstract

The invention provides a chromatin conformation capturing method of frozen tissue, and belongs to the technical field of three-dimensional genomics. According to the chromatin conformation capturing method of the frozen tissue, the scheme of double fixation and double enzyme digestion is adopted, an interaction matrix with the high resolution ratio can be obtained, finer close-range loops can be identified, and the false positive rate is lower. The mild lysis solution is used to ensure that permeation treatment is carried out on cell membranes, damage to internal chromatin structures of cell nucleuses is avoided, and signal noise is also reduced. A bridge linker with a biotin label is used for connection, and a Tn5 enzyme and a biotin enrichment mode are combined for final library construction, so that the connection efficiency is improved, the time required for library construction is shortened, and a special instrument is not needed. The chromatin conformation capturing method disclosed by the invention has the advantages of high resolution and high signal-to-noise ratio, and has great significance on three-dimensional genome research.

Description

Technical Field

[0001] The present invention belongs to the technical field of three-dimensional genomics, and in particular relates to a method for capturing chromatin conformation of frozen tissue. Background Art

[0002] With the completion of sequencing for the human genome, model plants and animals, and important crops and agricultural animal genomes, life science has entered a post-sequencing era centered on functional genomics. However, the mechanisms by which genomic information guides gene expression in specific spaces and times remain to be elucidated. In 2002, a proximity-linking-based chromatin conformation capture technique (3C) debuted, enabling the capture of single-site interactions. Circular chromatin conformation capture (4C) was developed in 2006, enabling the capture of single-site interactions across the entire genome. The same year, 5C (chromosome conformation capture carbon copy) technology was developed, enabling the capture of interactions between multiple regions.

[0003] With the advancement of sequencing technology, high-throughput chromatin conformation capture technology (Hi-C) was born in 2009 based on 3C technology, mapping the chromatin conformation of the entire genome for the first time. Hi-C is one of the necessary technologies for studying the spatial structure of the genome, analyzing the interactions between genes and regulatory elements, and thus clarifying the regulatory mechanism of gene expression changes. After more than ten years of development, Hi-C technology is currently divided into two categories: (1) Hi-C technology based on chromatin cleavage with sticky end endonucleases or nucleases, combined with biotin-labeled deoxynucleotides and blunt end linkers for blunt end ligation strategy, including dilute Hi-C, in situ Hi-C, DLO Hi-C, Hi-C3.0 using double sticky end restriction endonucleases DpnII and DdeI, and Micro-C; (2) BL-Hi-C using a single blunt end restriction endonuclease HeaIII combined with a two-step ligation method (blunt end addition A and bridge linker ligation).

[0004] Among these techniques, DLO Hi-C, Hi-C3.0, and Micro-C utilize blunt-end ligation strategies, resulting in lower ligation efficiency and a greater likelihood of random ligation compared to two-step ligation. Furthermore, previous studies have primarily used living cells, which cannot reflect the true chromatin conformation of cryopreserved samples. This has made the application of Hi-C technology difficult for cryopreserved samples, especially those stored at low temperatures for long periods. Summary of the Invention

[0005] The present invention provides a method for capturing chromatin conformation of frozen tissue, which can significantly improve the efficiency of capturing three-dimensional interactions between protein-mediated whole-genome DNA in animal frozen tissue samples.

[0006] The present invention provides a method for capturing chromatin conformation of frozen tissue, comprising the following steps:

[0007] (1) After the frozen tissue is crushed, double cross-linking is performed using formaldehyde solution and EGS solution. After the cross-linking reaction is terminated, the cross-linked tissue sample is separated and collected;

[0008] (2) using a lysis solution to lyse the tissue sample obtained in step (1) to obtain permeabilized tissue cells, and using two four-base blunt-end restriction endonucleases to jointly cut the permeabilized tissue cells to obtain enzyme cleavage products;

[0009] (3) performing an A addition reaction on the 3' end of the enzymatic cleavage product of step (2) to obtain an A addition product;

[0010] (4) mixing the A-added product of step (3) with a biotin-labeled bridge linker, performing proximity ligation to obtain a ligation product; decrosslinking the ligation product to obtain DNA, cleaving the obtained DNA using Tn5 transposase, and recovering the cleavage product;

[0011] (5) Capturing the DNA fragments with bridge linkers in the cleavage products obtained in step (4) by pre-blocked streptavidin-coated magnetic beads, and obtaining the final library by PCR amplification and product recovery;

[0012] (6) The library obtained in step (5) is quality controlled and subjected to high-throughput sequencing, and the three-dimensional results of the target tissue are analyzed.

[0013] In a preferred embodiment of the present invention, during the double cross-linking in step (1), the concentration of formaldehyde in the formaldehyde solution is 1% to 2% (v / v), and the final concentration of EGS in the EGS solution is 1.5 to 3 mM.

[0014] In a preferred embodiment of the present invention, the terminating cross-linking in step (1) comprises using glycine as a cross-linking terminator.

[0015] In a preferred embodiment of the present invention, the lysis in step (2) comprises sequentially performing a first lysis at 4° C. using a first lysis solution and performing a second lysis at room temperature using a second lysis solution;

[0016] The first lysis buffer includes 0.1% SDS FA lysis buffer; the 0.1% SDS FA cell lysis buffer includes the following components at the following concentrations: 50 mM Hepes-KOH, 150 mM NaCl, 1 mM EDTA, 1% (v / v) Triton-X-100, 0.1% (v / v) Sodium Deoxycholate and 0.1% (v / v) SDS;

[0017] The second lysing solution is a mixed solution containing SDS and BSA, wherein the final concentration of SDS is 0.1% (v / v) and the final concentration of BSA is 0.5% (v / v).

[0018] In a preferred embodiment of the present invention, the four-base blunt-end restriction endonuclease in step (2) includes Alu1 and Hael1.

[0019] In a preferred embodiment of the present invention, the nucleotide sequence of the forward strand of the bridge linker in step (4) is shown as SEQ ID No. 1 or SEQ ID No. 3, and the nucleotide sequence of the antisense strand is shown as SEQ ID No. 2 or SEQ ID No. 4.

[0020] In a preferred embodiment of the present invention, the method for preparing the pre-blocked streptavidin-coated magnetic beads in step (5) comprises sequentially treating the streptavidin-coated magnetic beads with the following steps: rinsing with 2×BWbuffer, shaking treatment with i-Blockbuffer, rinsing with 1×BWbuffer, shaking treatment with zebrafish genomic DNA, and rinsing with 1×BWbuffer.

[0021] In a preferred embodiment of the present invention, the PCR amplification procedure in step (5) includes: heating the cover at 105°C; gap filling at 72°C for 3 minutes; pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 15 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds, for 8 to 9 cycles; extension at 72°C for 5 minutes; and storage at 4°C.

[0022] In a preferred embodiment of the present invention, the primer pair used for PCR amplification in step (5) is a dedicated primer pair designed based on the Illumina sequence platform.

[0023] In a preferred embodiment of the present invention, the data analysis in step (6) includes: processing the original sequencing data using the adapter sequence information to remove the adapters to obtain pure adapter-free sequencing data;

[0024] According to the location of the linker on the genome, the sequencing data without the linker are divided into: sequencing data with linkers at both ends, sequencing data with linkers at only one end, and sequencing data without linkers at both ends;

[0025] The sequencing data with and without linkers at both ends are aligned to the reference genome to obtain non-redundant alignment results; the restriction site information on the reference genome is used to determine the valid interaction pairs with and without linkers at both ends using the alignment results, and all valid interaction pairs are integrated to construct the final interaction matrix.

[0026] Beneficial effects: The present invention provides a method for capturing chromatin conformation of frozen tissues, which adopts a double fixation and double enzyme digestion scheme, can obtain a higher-resolution interaction matrix, identify more delicate close-range loops, and has lower false positives. The use of a mild lysis solution ensures that the cell membrane is permeabilized while avoiding damage to the chromatin structure inside the cell nucleus, and also reduces signal noise. The use of a biotin-labeled bridge linker for connection, combined with Tn5 enzyme and biotin enrichment for the final library construction, improves the connection efficiency, reduces the time required for library construction, and does not require special instruments. It is of great significance for three-dimensional genome research. The chromatin conformation capture method of the present invention has the advantages of high resolution and high signal-to-noise ratio. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the experimental principle of the chromatin conformation capture method for frozen tissues of the present invention;

[0028] Figure 2 Flowchart for data analysis;

[0029] Figure 3 is the size distribution of DNA fragments after double enzyme digestion;

[0030] Figure 4 is the size distribution of DNA fragments after ligation;

[0031] Figure 5 This is the distribution diagram of DNA fragments after TTE digestion;

[0032] Figure 6 is the size distribution of DNA fragments in the library;

[0033] Figure 7 This is the interaction heat map obtained in Example 1 of the present invention;

[0034] Figure 8 This is the interaction heat map obtained in Comparative Example 1;

[0035] Figure 9 Density curve of the number of reads for loops;

[0036] Figure 10 It is the distance density curve of loops;

[0037] Figure 11 1 is a graph comparing the intensity of the method of Example 1 of the present invention and in-suit Hi-C loops. The method in the figure represents the method of Example 1. DETAILED DESCRIPTION

[0038] The present invention provides a method for capturing chromatin conformation of frozen tissue. The experimental principle is as follows: Figure 1 As shown, the following steps are included:

[0039] (1) After the frozen tissue is pulverized, double cross-linking is performed using formaldehyde solution (Crossing-linking buffer) and EGS solution (EGS buffer). After terminating the cross-linking reaction, the cross-linked tissue sample is collected by centrifugation;

[0040] (2) using a lysis solution to lyse the tissue sample obtained in step (1) to obtain permeabilized tissue cells, and using two four-base blunt-end restriction endonucleases to jointly cut the permeabilized tissue cells to obtain enzyme cleavage products;

[0041] (3) performing an A addition reaction on the 3' end of the enzymatic cleavage product of step (2) to obtain an A addition product;

[0042] (4) mixing the A-added product of step (3) with a biotin-labeled bridge linker to obtain a ligation product; decrosslinking the ligation product to obtain DNA, cleaving the obtained DNA using Tn5 transposase, and recovering the cleavage product;

[0043] (5) Capturing the DNA fragments with bridge linkers in the cleavage products obtained in step (4) by pre-blocked streptavidin-coated magnetic beads, and obtaining the final library by PCR amplification and product recovery;

[0044] (6) The library obtained in step (5) is quality controlled and subjected to high-throughput sequencing, and the three-dimensional results of the target tissue are analyzed.

[0045] The present invention is directed to frozen animal tissue samples, and there is no particular limitation on the freezing time. The present invention involves cryogenically grinding the frozen animal tissue sample after removal, with the mortar used pre-cooled in liquid nitrogen before cryogenic grinding.

[0046] In the present invention, cryogenically ground tissue powder is double-crosslinked using formaldehyde and EGS. The formaldehyde concentration in the formaldehyde solution is 1% to 2% (v / v), and the EGS concentration in the EGS solution is 1.5 to 3 mM. The double crosslinking method of the present invention involves first crosslinking with formaldehyde, then terminating the crosslinking with glycine, then crosslinking with EGS, and then terminating the crosslinking with glycine. The crosslinking is performed at room temperature, with formaldehyde crosslinking for 10 to 30 minutes, and in one embodiment, 20 minutes; and EGS crosslinking for 1 to 2 hours, and in one embodiment, 1 hour.

[0047] After terminating cross-linking, the cross-linked tissue sample is centrifuged to obtain a cross-linked tissue sample. In one embodiment, the centrifugation is performed at 2000 g for 5 minutes at a low temperature of 4°C. The cross-linked tissue sample can also be washed with PBST (0.1% Triton-X-100) to remove the cross-linking agent. In one embodiment, after washing, the sample is divided into 1 to 6 1.5 mL EP tubes, each containing 0.05 g.

[0048] Take an EP tube containing a tissue sample and use a mild lysis solution to lyse it to obtain permeable tissue cells. The lysis of the present invention includes sequentially using a first lysis solution at 4°C for a first lysis and a second lysis solution at room temperature for a second lysis. In one embodiment, the first lysis solution includes 0.1% SDS FA lysis buffer; the 0.1% SDS FA cell lysis buffer includes the following components at the following concentrations: 50mM Hepes-KOH, 150mM NaCl, 1mM EDTA, 1% (v / v) Triton-X-100, 0.1% (v / v) Sodium Deoxycholate and 0.1% (v / v) SDS; the second lysis solution is a mixture containing SDS and BSA (0.1% SDS buffer), wherein the final concentration of SDS is 0.1% (v / v) and the final concentration of BSA is 0.5% (v / v); wherein the time for the first lysis is 1 hour and the time for the second lysis is 10 minutes. The present invention terminates the lysis after the lysis, and the termination of the lysis includes the use of a termination buffer, which is a mixture containing Triton-X-100 and BSA, wherein the final concentration of Triton-X-100 is 2% (v / v), and the final concentration of BSA is 0.5% (v / v). The temperature for terminating the lysis according to the present invention can be 37°C, and it can be left to stand for 10 minutes. The present invention performs centrifugation after terminating the lysis and discards part of the liquid. The present invention uses a milder tissue lysis method to ensure that the cell membrane is permeabilized while avoiding damage to the chromatin structure inside the cell nucleus, while reducing signal noise.

[0049] The present invention uses a four-base blunt-end restriction endonuclease to perform double enzyme digestion on the permeabilized tissue cells. After the double enzyme digestion, blunt-end DNA fragments can be obtained. In one embodiment, the four-base blunt-end restriction endonucleases described in the present invention are Alu1 and Haelll. The double enzyme digestion cutting system described in the present invention is measured in 500 μL and includes: lysate, NEB 10×cutsmart 50 μL, 50× protease inhibitor 10 μL, 10% high molecular weight Triton-X-100 50 μL, Alu1 restriction endonuclease 25 μL, Haelll restriction endonuclease 15 μL and 350 μL of water. The parameters of the double enzyme digestion described in the present invention, in one embodiment, are enzyme digestion with shaking at 37°C for 4 to 6 hours. The present invention uses Alu1 and hael1 four-base restriction endonucleases for enzyme digestion, so that the genome is fully broken and subsequently fully connected, thereby improving the interaction resolution and being able to detect closer and finer loops. The identified loops have more reads counts, resulting in fewer false positives.

[0050] The present invention performs an A addition operation on the obtained double-enzyme digestion product to obtain an A-added product. In one embodiment of the present invention, the A addition operation is performed on the 3' end of the blunt-end DNA fragment obtained by the double-enzyme digestion. The reaction system for the A addition operation, calculated in 500 μL, includes: a base reaction system comprising: enzyme digestion product, 50 μL of 5% BSA, 10 μL of 10 mM dATP, 250 μL of 10×NEB buffer, 10 μL of Klenow 3'→5' enzyme, and 330 μL of water. In one embodiment, the A addition operation of the present invention is performed at 37° C. with shaking for 1 to 2 hours.

[0051] The present invention mixes the A-added product with a biotin-labeled bridge linker, and performs a ligation reaction on adjacent interacting DNA to obtain a ligation product. The nucleotide sequence of the forward strand of the bridge linker is shown in SEQ ID No. 1 or SEQ ID No. 3, and the nucleotide sequence of the antisense strand is shown in SEQ ID No. 2 or SEQ ID No. 4. The ligation is performed under the action of T4 DNA ligase. The enzyme ligation system per mL includes: A-added product, 100 μL of 10% Triton-X-100, 200 μL of NEB 5× quick ligase buffer, 4 μL of bridge-linker (200 ng / μL), 10 μL of 50× protease inhibitor, 100 μL of 5% BSA, 10 μL of T4 DNA ligase (400 U / ml), and the balance of water. In one embodiment, the enzyme ligation system is shaken at 16° C. for 20 hours.

[0052] Sense strand (SEQ ID No. 1): 5′- / 5Phos / CGCGATATC / iBIOdT / TATCGACT-3′;

[0053] Antisense strand (SEQ ID No. 2): 5'- / 5Phos / GTCAGATAAGATATCGCGT-3';

[0054] or sense strand (SEQ ID No. 3): 5′- / 5Phos / CGCGATATCTAG / iBIOdT / ACGACT-3;

[0055] Antisense strand (SEQ ID No. 4): 5'- / 5Phos / GTCGTACTAGATATCGCGT-3'.

[0056] The present invention decrosslinks the ligation product to obtain DNA, extracts a portion of the DNA, and uses Tn5 transposase to cleave the DNA at 55°C for 10 minutes. The resulting product is purified and recovered using a DNA purification kit to obtain a cleavage product. The present invention uses Tn5 enzyme to fragment the DNA and insert the primer template strand for PCR amplification, reducing the time required for library construction and eliminating the need for special equipment.

[0057] The present invention uses pre-blocked streptavidin-coated magnetic beads to capture DNA fragments with bridgelinkers in the cleavage product, wherein the streptavidin magnetic beads are first blocked, rinsed twice with 2×BW buffer, then treated with i-Block buffer at 25°C with shaking for 45 minutes, rinsed once with 1×BW buffer, then treated with zebrafish genomic DNA at 25°C with shaking for 30 minutes, and then rinsed once with 1×BW buffer. The present invention mixes the streptavidin-coated magnetic beads that have undergone the blocking treatment with the cleavage product, shakes at 25°C for 1 hour, washes three times with 2×SSC+0.5% SDS, each time with shaking at 37°C for 5 minutes; then washes twice with 1×BW buffer, each time with shaking at 37°C for 3 minutes; then rinses once with TE, discards the supernatant, and resuspends the magnetic beads with TE.

[0058] The present invention obtains a final library through PCR amplification and product recovery, wherein the primers used for PCR amplification are synthesized based on primers specifically for the Illumina sequencing platform. In one embodiment, primers P5 and P7 are used for PCR amplification. The PCR amplification procedure includes: 105°C heated lid; 72°C gap filling for 3 minutes; 98°C pre-denaturation for 30 seconds; 98°C denaturation for 15 seconds, 60°C annealing for 30 seconds, 72°C extension for 30 seconds, 8-9 cycles; 72°C extension for 5 minutes; and storage at 4°C. The primer pair P5 and P7 used in one embodiment of the present invention was purchased from Novation Biosciences, catalog number TD203.

[0059] The present invention controls the quality of the obtained library, performs high-throughput sequencing, and analyzes the three-dimensional results of the target tissue. The data analysis process is as follows: Figure 2 As shown, the raw sequencing data is first processed through the linker sequence information to remove the linkers to obtain pure linker-free sequencing data. Subsequently, according to the position of the linker on the genome, the linker-free sequencing data is classified into three categories: sequencing data with linkers at both ends, sequencing data with linkers at only one end, and sequencing data without linkers at both ends. Next, the sequencing data with and without linkers at both ends will be aligned to the reference genome to obtain non-redundant alignment results. Using the enzyme cutting site information on the reference genome, these alignment results are further used to determine the effective interaction pairs with and without linkers at both ends. Finally, all effective interaction pairs are integrated to construct the final interaction matrix.

[0060] To further illustrate the present invention, a method for capturing chromatin conformation of frozen tissue provided by the present invention is described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0061] In the present invention, unless otherwise specified, room temperature refers to 25°C.

[0062] The instruments, equipment, and reagents used in the implementation of the present invention are all commercially available to those skilled in the art. Unless otherwise specified, the technical means used in the implementation of the present invention are conventional means well known in the art.

[0063] Solution preparation:

[0064] Crossing-linking buffer: Contains 0.1 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM Hepes (pH 8.0), ddH2O, and 1% unimolecular formaldehyde. Prepare and use immediately.

[0065] EGS buffer: Weigh 4.575 mg of EGS, dissolve in 25 μL of DMSO, and add to 5 mL of PBS preheated at 37°C.

[0066] 0.1% SDS FAcell lysis buffer: contains 50Mm Hepes-KOHpH7.5, 150mM NaCl, 1mMEDTA, 1% Triton-X-100, 0.1% Sodium Deoxycholate, 0.1% SDS and ddH2O;

[0067] 0.1% SDS buffer: contains 0.1% SDS, 0.5% BSA, 1× protease inhibitor, and ddH2O. Prepared for immediate use.

[0068] Stop buffer: contains 2% Triton-X-100, 0.5% BSA, and ddH2O;

[0069] Enzyme digestion buffer: contains 1× cutsmart, 1× protease inhibitor, 1% Triton-X-100 and ddH2O, prepared for immediate use;

[0070] A-Tailing buffer: contains 0.5% BSA, 0.2 mM dATP, 1% Triton-X-100, 1× NEB buffer 2, and ddH2O. Prepared for use immediately.

[0071] Linker buffer: contains 800 ng Bridge linker, 0.5% BSA, 1% Triton-X-100, 1× protease inhibitor, 5× quick ligase buffer, 1× protease inhibitor, and ddH2O;

[0072] Elution buffer: contains 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% SDS, and ddH2O;

[0073] 2× BW buffer: contains 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 M NaCl, 0.01% Tween 20, and ddH2O;

[0074] 1×BWbuffer: dilute 2×BWbuffer with ddH2O;

[0075] iBlock buffer: Contains 0.02g / mL Thermo iBlock, 0.5% SDS, 20mM Tris-HCl (pH 7.5), 137mM NaCl, and ddH2O.

[0076] Example 1

[0077] 1. Grind and weigh tissue samples

[0078] (1) Take liver tissue samples from 180-day-old Large White pigs frozen in a -80°C refrigerator;

[0079] (2) Use liquid nitrogen to pre-cool the mortar and pestle, grind the sample for 15 min until it is completely powdered, and then put it into a tube;

[0080] (3) Accurately weigh 0.05–0.5 g of ground tissue sample and place it in a 15 mL centrifuge tube.

[0081] 2. Tissue Fixation

[0082] (1) Place the weighed sample on ice and resuspend in 5 mL of PBS;

[0083] (2) Add 500 μL cross-linking buffer and fix at room temperature for 40 min;

[0084] (3) Add 275 μL of 2.5 M glycine to terminate cross-linking for 10 min;

[0085] (4) Centrifuge at 2000 g for 5 min at 4°C and discard the supernatant;

[0086] (5) Add 5 mL of EGS buffer and fix at room temperature for 1 h;

[0087] (6) Add 400 μL of 2.5 M glycine to terminate cross-linking for 10 min;

[0088] (7) Centrifuge at 2000 g for 5 min at 4°C and discard the supernatant;

[0089] (8) Resuspend in PBS and dispense into 1.5 mL EP tubes (0.1 g sample per tube);

[0090] (9) Centrifuge at 2000 g for 5 min at 4°C and discard the supernatant;

[0091] (10) Quickly freeze in liquid nitrogen and store in a -80°C freezer.

[0092] 3. Tissue Lysis

[0093] (1) Add 1 ml of cold PBST (0.01% Triton-X-100) to the fixed sample;

[0094] (2) Centrifuge at 2000 rcf at 4°C for 5 min and discard the supernatant;

[0095] (3) Add 1 mL of 0.1% SDS-Fluorolysis buffer and lyse at 4°C for 1 h;

[0096] (4) Centrifuge at 2000 rcf at 4°C for 5 min and discard the supernatant;

[0097] (5) Add 200 μL of 0.1% SDS buffer and lyse at room temperature for 10 min;

[0098] (6) Add 200 μL stop buffer, mix well, and incubate at 37°C for 10 min;

[0099] (7) Mix again, centrifuge at 1000 rcf at 4°C for 5 min, and discard the supernatant.

[0100] 4. Enzyme digestion reaction

[0101] (1) Add 460 μL of enzyme digestion buffer, then add 25 μL of Alu1 enzyme (NEB, R0137L) and 15 μL of Haelll enzyme (NEB, R0108L), invert to mix, and digest at 37°C for 4-6 h;

[0102] (2) Centrifuge at 1000 rcf at 4°C for 5 min and discard the supernatant;

[0103] (3) Add 1 ml of cold PBST (0.01% Triton-X-100), take 10 μL of the solution into a new 1.5 mL EP tube, extract the DNA, run the enzyme digestion on an Agilent 5200 instrument, and check the enzyme digestion status. Centrifuge at 1000 rcf at 4°C for 5 min and discard the supernatant.

[0104] The results are as follows Figure 3 As shown, the genomic fragments have been cut into appropriate sizes, and there are still large fragments of more than 10,000 bp, which proves that the lysis method used in the present invention is relatively mild and ensures the integrity of the cell nucleus.

[0105] 5. Adding A base to the end of DNA fragment

[0106] (1) Add 490 μL of A-Tailing buffer to the digested tissue, then add 10 μL of Klenow enzyme and react at 37°C for 1 to 2 hours.

[0107] (2) Centrifuge at 1000 rcf at 4°C for 5 min and discard the supernatant; add 1 ml of cold PBST (0.01% Triton-X-100) to wash once, centrifuge at 1000 rcf at 4°C for 5 min and discard the supernatant.

[0108] 6. Proximity Connection

[0109] Add 990 μL linker buffer to the tissue after adding A base, then add 10 μL T4 DNA ligase, and react at 16°C for 20 hours.

[0110] After extracting DNA, run the Agilent 5200 instrument and check the connection status.

[0111] The results are as follows Figure 4 As shown, the size of the DNA fragment is larger than that of the fragment after enzyme digestion, which proves that the connection effect is good.

[0112] 7. DNA extraction

[0113] (1) Centrifuge at 1000 rcf at 4°C for 5 min and discard the supernatant;

[0114] (2) Add 1 mL of PBST (0.1% Triton-X-100);

[0115] (3) Centrifuge at 1000 rcf at 4°C for 5 min and discard the supernatant;

[0116] (4) Add 150 μL Elution buffer and incubate at 65°C, 13,000 rpm for 30 min to decrosslink.

[0117] (5) Add 250 μL TE and 16 μL proteinase K and digest at 55°C and 650 rpm for 8 h.

[0118] 8. Tn-5 enzyme digestion

[0119] (1) Test TTE enzyme amount: 30 μL system, including 50 ng DNA, 6 μL 5× TTBL, TTE enzyme and ddH2O;

[0120] (2) Run Agilent 5200 to check the fragment distribution. Determine the amount of enzyme, the result is as follows Figure 5 As shown, when the fragments are mainly distributed between 200-300 bp, the amount of TTE enzyme is appropriate. You can proceed to the next step;

[0121] (3) After determining the amount of enzyme, a total of 300 ng was cut;

[0122] (4) Recover DNA using a kit.

[0123] 9. Streptavidin magnetic bead capture

[0124] (1) Take 25 μL of streptavidin magnetic beads, place them in a low-binding EP tube, place them on a magnetic stand, and discard the liquid after it has clarified.

[0125] (2) Add 500 μL of 2× BW buffer, mix thoroughly by inversion, place on a magnetic stand, and discard the liquid after the liquid is clarified. Repeat twice;

[0126] (3) Add 300 μL iBlock buffer and block the magnetic beads at room temperature for 45 minutes;

[0127] (4) Place on a magnetic rack and discard the liquid after it clarifies;

[0128] (5) Add 500 μL of 1× BW buffer, mix thoroughly by inversion, place on a magnetic stand, and discard the liquid after it clarifies;

[0129] (6) Add 200 μL 2×BW buffer, 195 μL ddH2O, and 5 μL zebrafish genomic DNA (100 ng / μL) and block at room temperature for 30 min;

[0130] (7) Place on a magnetic rack and discard the liquid after it clarifies;

[0131] (8) Add 500 μL of 1× BW buffer, mix thoroughly by inversion, place on a magnetic stand, and discard the liquid after the liquid has clarified. Repeat twice;

[0132] (9) Add ddH2O to the DNA recovered in step 8-(3) to make up to 200 μL, then add 200 μL 2×BW buffer and transfer to the EP tube treated in step 9-(8) and capture at room temperature for 1 h.

[0133] 10. Wash the magnetic beads

[0134] (1) Add 500 μL of 0.5% SDS, 2× SSC, and ddH2O, treat at 37°C for 5 min, centrifuge gently, place on a magnetic stand, and discard the liquid after the liquid is clarified. Repeat three times;

[0135] (2) Add 500 μL of 1× BW buffer, treat at 37°C for 3 min, centrifuge gently, place on a magnetic stand, discard the liquid after the liquid is clarified, and repeat twice;

[0136] (3) Add 1 mL of 1×TE, mix thoroughly by inverting, centrifuge gently, place on a magnetic rack, and discard the liquid after it clarifies.

[0137] (4) Add 30 μL of 1×TE and mix well.

[0138] 11. PCR amplification

[0139] (1) Take 200 μL of EP tube without DNAase and RNAase, and prepare 50 μL reaction system: 10 μL of purified DNA fragment, 5 μL of Primer P5 (10 μM), 5 μL of Primer P7 (10 μM), 5 μL of PPM, Add 25 μL of High-Fidelity 2X PCR Master Mix. Pipette the reaction mixture 20 times to mix thoroughly, then centrifuge briefly.

[0140] (2) The obtained DNA was amplified in three tubes. The PCR amplification conditions were as follows: 105°C hot cover; 72°C gap filling for 3 min; 98°C pre-denaturation for 30 s; 98°C denaturation for 15 s, 60°C annealing for 30 s, 72°C extension for 30 s, 8-9 cycles; 72°C extension for 5 min; and storage at 4°C.

[0141] 12. Library recovery and purification

[0142] (1) 0.55× beads screened out fragments less than 800 bp, and 0.3× beads screened out fragments greater than 200 bp.

[0143] (2) The library concentration was determined by Qubit, and the fragment size distribution of the library was detected by Agilent 2100 Bioanalyzer.

[0144] (3) According to the quality control of the library, 1.1× DNA was used to remove excess primers and the concentration was determined by Qbit. Figure 6 As shown, the final library fragment size on the machine is 200 to 800 bp.

[0145] 13. Sequencing

[0146] 14. Data Analysis

[0147] (1) The raw sequencing data is first processed using the adapter sequence information to remove the adapters and obtain pure adapter-free sequencing data.

[0148] (2) According to the location of the linkers on the genome, the sequencing data without linkers are classified into three categories: sequencing data with linkers at both ends, sequencing data with linkers at only one end, and sequencing data without linkers at both ends.

[0149] (3) Sequencing data with and without linkers at both ends will be aligned to the reference genome SusScr11 to obtain non-redundant alignment results.

[0150] (4) Using the restriction enzyme site information on the reference genome, these alignment results are further used to determine valid interaction pairs with and without linkers at both ends. Ultimately, all valid interaction pairs are integrated to construct the final interaction matrix.

[0151] Comparative Example 1

[0152] With reference to the method described in the article “A 3D Map of the Human Genome at Kilobase Resolution Reveals Principles of Chromatin Looping” (DOI: 10.1016 / j.cell.2014.11.021), the same samples as in Example 1 were detected using in situ Hi-C.

[0153] The results of Example 1 and Comparative Example 1 are shown in Table 1. Figure 7 and Figure 8 As shown, among the effective interaction fragments obtained, the ratio of close-range interactions obtained by the method described in Example 1 is higher than that in suit Hi-C, proving that the method described in the present invention is better than in suit Hi-C.

[0154] Table 1 Effective interaction fragment data of Example 1 and Comparative Example 1

[0155]

[0156]

[0157] Compared with in-suit Hi-C, the number of reads of loops identified by this invention is greater ( Figure 9 ), indicating that the loops identified by the present invention are more accurate and have lower false positives; compared with in suit Hi-C, the loops identified by the method of the present invention are more inclined to close-range loops ( Figure 10 ), indicating that the method can identify loops more precisely; compared on the Hi-C heat map, the loops identified by the method of the present invention contain more read counts than the loops identified by in suit Hi-C ( Figure 11 ).

[0158] At the same time, the costs in Example 1 and Comparative Example 1 were statistically analyzed, and the results are shown in Table 2. The method described in Example 1 has lower costs.

[0159] Table 2 Cost difference between Example 1 and in-suit Hi-C

[0160]

[0161]

[0162] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for capturing chromatin conformation of frozen tissue, characterized in that: The following steps are involved: (1) After the frozen tissue is crushed, double cross-linking is performed using formaldehyde solution and EGS solution. After the cross-linking reaction is terminated, separation is performed and the cross-linked tissue sample is collected; (2) using a lysis solution to lyse the tissue sample obtained in step (1) to obtain permeabilized tissue cells, and using two four-base blunt-end restriction endonucleases to jointly cut the permeabilized tissue cells to obtain enzyme cleavage products; (3) performing an A addition reaction on the 3' end of the enzymatic cleavage product of step (2) to obtain an A addition product; (4) mixing the A-added product of step (3) with a biotin-labeled bridge linker, performing proximity ligation to obtain a ligation product; decrosslinking the ligation product to obtain DNA, cleaving the obtained DNA using Tn5 transposase, and recovering the cleavage product; (5) Capturing the DNA fragments with bridge linkers in the cleavage products obtained in step (4) by pre-blocked streptavidin-coated magnetic beads, and obtaining the final library by PCR amplification and product recovery; (6) The library obtained in step (5) is quality controlled and subjected to high-throughput sequencing, and the three-dimensional results of the target tissue are analyzed.

2. The chromatin conformation capture method according to claim 1, characterized in that During the double cross-linking in step (1), the volume percentage of formaldehyde in the formaldehyde solution is 1% to 2%, and the concentration of EGS in the EGS solution is 1.5 to 3 mM.

3. The chromatin conformation capture method according to claim 1, characterized in that The cross-linking terminator used in step (1) to terminate the cross-linking includes glycine.

4. The chromatin conformation capture method according to claim 1, characterized in that The lysis in step (2) comprises sequentially performing a first lysis at 4° C. using a first lysis solution and performing a second lysis at room temperature using a second lysis solution; The first lysis buffer includes 0.1% SDS FA lysis buffer; the 0.1% SDS FA cell lysis buffer includes the following components at the following concentrations: 50 mM Hepes-KOH, 150 mM NaCl, 1 mM EDTA, 1% (v / v) Triton-X-100, 0.1% (v / v) Sodium Deoxycholate and 0.1% (v / v) SDS; The second lysing solution is a mixed solution containing SDS and BSA, wherein the final concentration of SDS is 0.1% (v / v) and the final concentration of BSA is 0.5% (v / v).

5. The chromatin conformation capture method according to claim 1, characterized in that The four-base blunt-end restriction endonuclease in step (2) includes Alu1 and Hael11.

6. The chromatin conformation capture method according to claim 1, characterized in that The nucleotide sequence of the forward strand of the bridgelinker in step (4) is shown as SEQ ID No. 1 or SEQ ID No. 3, and the nucleotide sequence of the antisense strand is shown as SEQ ID No. 2 or SEQ ID No.

4.

7. The chromatin conformation capture method according to claim 1, characterized in that The preparation method of the streptavidin-coated magnetic beads pre-blocked in step (5) comprises treating the streptavidin-coated magnetic beads in sequence by the following steps: rinsing with 2×BW buffer, shaking treatment with i-Block buffer, rinsing with 1×BW buffer, shaking treatment with zebrafish genomic DNA, and rinsing with 1×BW buffer.

8. The chromatin conformation capture method according to claim 1 or 7, characterized in that The PCR amplification procedure in step (5) includes: 105°C hot cover; 72°C gap filling for 3 minutes; 98°C pre-denaturation for 30 seconds; 98°C denaturation for 15 seconds, 60°C annealing for 30 seconds, 72°C extension for 30 seconds, 8 to 9 cycles; 72°C extension for 5 minutes; and storage at 4°C.

9. The chromatin conformation capture method according to claim 8, characterized in that The primer pair used for the PCR amplification in step (5) is a special primer pair designed based on the Illumina sequence platform.

10. The chromatin conformation capture method according to claim 1, characterized in that The data analysis in step (6) includes: processing the original sequencing data using the adapter sequence information to remove the adapters to obtain pure adapter-free sequencing data; According to the location of the linker on the genome, the sequencing data without the linker are divided into: sequencing data with linkers at both ends, sequencing data with linkers at only one end, and sequencing data without linkers at both ends; The sequencing data with and without linkers at both ends are aligned to the reference genome to obtain non-redundant alignment results; the restriction site information on the reference genome is used to determine the valid interaction pairs with and without linkers at both ends using the alignment results, and all valid interaction pairs are integrated to construct the final interaction matrix.

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