Method for multi-dimensional analysis of cell epigenomics

Abstract

The application discloses a method for analyzing cell epigenomics in multiple dimensions, which comprises the following steps: embedding different linker sequences in cells by using ChiTag transposase and conventional Tn5 transposase respectively, and realizing the joint analysis of chromatin open region information and specific protein binding sequence information at the cell level. The method has important application prospects in the research of cell population heterogeneity related to development and / or diseases, the drawing of cell atlas, the analysis of tumor cells with different clinical characteristics, the clinical research of tumor cell evolution and / or metastasis and the like.

Description

Technical Field

[0001] The present invention relates to the field of bioinformatics, and in particular to a method for multi-dimensional analysis of cellular epigenomics. Background Art

[0002] Epigenomics technology is an important tool for analyzing epigenetics. Compared with technologies that can only describe the average epigenomic characteristics of the entire cell population, epigenomic methods are becoming a new technology for studying cellular plasticity and heterogeneity. Currently, many epigenomic technologies have been developed to describe the epigenome of heterogeneous tumor tissues at the single-cell and multi-cell levels.

[0003] The CUT&Tag technology uses ChiTag enzyme to specifically break the DNA near the target protein and connect it to the adapter. Then, the adapter sequence on the breaking enzyme is used for amplification to obtain a specific protein interaction DNA library for high-throughput sequencing. Figure 1 , reference: https: / / doi.org / 10.1038 / s41467-019-09982-5.

[0004] ATAC-seq technology uses conventional Tn5 transposase to break the DNA in the open region of the chromosome and connect it to the adapter. Then, the adapter sequence on the breaking enzyme is used for amplification to obtain the DNA library of the open region of the chromosome for high-throughput sequencing. Figure 2 , reference: doi:10.1002 / 0471142727.mb2129s109.

[0005] At present, although both CUT&Tag and ATAC-seq technologies are relatively mature, neither technology can jointly analyze the specific protein interaction DNA information and chromosome open region information of the same cell.

[0006] Invention disclosure

[0007] The problems to be solved by the present invention are: 1) to simultaneously capture the DNA sequence information of specific protein interactions and the information of chromosome open regions; 2) to achieve high-throughput library construction and sequencing using the iDrop platform.

[0008] In a first aspect, the present invention claims a method for multi-dimensional analysis of cellular epigenomics.

[0009] The method for multi-dimensional analysis of cellular epigenomics claimed in the present invention may include the following steps: using ChiTag transposase (a fusion protein of Protein A protein and Tn5 transposase) and conventional Tn5 transposase (only Tn5 transposase) in cells to embed different linker sequences respectively, so as to achieve the joint analysis of chromatin open region information and specific protein binding sequence information at the cellular level.

[0010] The method may be method A or method B.

[0011] Method A is a method for multi-dimensional analysis of single-cell epigenomics, which may specifically include the following steps:

[0012] (A1) Change the permeability of the test cells;

[0013] (A2) adding an antibody (primary antibody) corresponding to a specific protein to the cells treated in step (A1) and incubating the cells;

[0014] (A3) adding a secondary antibody (antibody against the antibody corresponding to the specific protein) to the cells treated in step (A2) for incubation;

[0015] (A4) adding ChiTag transposase to the cells treated in step (A3) and incubating;

[0016] (A5) Adding reaction reagents to the cells treated in step (A4) for incubation, and then adding conventional Tn5 transposase; this step achieves DNA fragmentation.

[0017] (A6) After the reaction in step (A5), single-cell droplets are generated using a water-in-oil structure (e.g., using a DNBelab C4 portable single-cell system) for intra-droplet amplification;

[0018] (A7) breaking the emulsion and performing amplification and purification;

[0019] (A8) performing enzyme digestion to obtain the final library;

[0020] (A9) Sequencing the final library obtained in step (A8) to analyze the chromatin open region information and the specific protein binding sequence information at the single-cell level.

[0021] Method B is a method for multi-dimensional analysis of multi-cellular epigenomics, which may specifically include the following steps:

[0022] (B1) Change the permeability of the test cells;

[0023] (B2) adding antibodies corresponding to specific proteins to the cells treated in step (B1) and incubating them;

[0024] (B3) adding a secondary antibody to the cells treated in step (B2) for incubation;

[0025] (B4) adding ChiTag transposase to the cells treated in step (B3) and incubating;

[0026] (B5) adding a reaction reagent to the cells treated in step (B4) for incubation, and then adding conventional Tn5 transposase;

[0027] (B6) amplification and purification;

[0028] (B7) performing enzyme digestion to obtain the final library;

[0029] (B8) High-throughput sequencing is performed on the final library obtained in step (B7) to analyze the chromatin open region information and specific protein binding sequence information at the multicellular level.

[0030] In steps (A1) and (B1) of the method, prior to altering the permeability of the test cells, the test cells may be pretreated as follows: collecting the test cells, centrifuging at 600 g for 3 minutes, and discarding the supernatant; resuspending the cells in at least one volume of basic wash buffer, centrifuging at 600 g for 3 minutes, and discarding the supernatant. All operations are performed at room temperature (23-28°C, the same below) to minimize stress on the cells.

[0031] In steps (A1) and (B1) of the method, the changing of the permeability of the test cells can be achieved by resuspending the test cells in NP40-digoxigenin washing buffer.

[0032] Furthermore, (A1) and (B1) include: first resuspending 500,000 to 1,000,000 cells in 1 ml of the NP40-digoxigenin wash buffer (i.e., the first resuspension), then centrifuging at 600 g for 3 minutes, discarding the supernatant, and adding 49 μl of the NP40-digoxigenin wash buffer supplemented with 1.5-2.5 mM (e.g., 2 mM) EDTA and resuspending the cells (i.e., the second resuspension).

[0033] The NP40-Digitonin Wash Buffer is obtained by adding 0.01% (volume percentage) NP40 and 0.01% digoxin to the basic wash buffer.

[0034] In a specific embodiment of the present application, the digoxin is BN2006 # Digitonin (5%) / brand & Invitrogen / size & 1, which weigt-% is 5. The concentration of digoxin in the NP40-digoxin wash buffer is equivalent to the concentration of digoxin in the product after 500-fold volume dilution.

[0035] The basic wash buffer described above is composed of: 20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM spermidine; 1x protease inhibitor cocktail (Sigma-Aldrich, Cat. No. 11873580001).

[0036] In steps (A2) and (B2) of the method, the specific protein corresponding antibody (primary antibody) is directly added to the second cell resuspension obtained in steps (A1) and (B1).

[0037] The ratio of adding the primary antibody in this step is about: 1 μL of the primary antibody is added to every 49 μL of the cell resuspension.

[0038] In a specific embodiment of the present application, in steps (A2) and (B2), the specific protein corresponding antibody (primary antibody) is H3K27me3 antibody. Correspondingly, the secondary antibody is the antibody against H3K27me3 antibody.

[0039] In steps (A2) and (B2) of the method, the incubation can be room temperature incubation for 2 h or 4°C incubation overnight (10-12 h, same below).

[0040] In steps (A3) and (B3) of the method, before adding the secondary antibody, a step of washing and centrifugation can also be included; the washing can be washing with the NP40-digoxin wash buffer described above; the centrifugation can be 600 g centrifugation for 3 min.

[0041] In a specific embodiment of the present application, the centrifugation and the washing are performed in turn and alternately, and there is one washing and one centrifugation in total; after centrifugation, the supernatant is discarded.

[0042] After centrifugation, a step of adding the NP40-digoxin wash buffer described above to resuspend the cells in the precipitate is also included.

[0043] The resuspension in this step can be about 50-100 million cells resuspended in 98 μL of the aforementioned NP40-Digitonin Wash Buffer.

[0044] In steps (A3) and (B3) of the method, the secondary antibody can be added to the cell suspension obtained in this step.

[0045] The secondary antibody can be added at a ratio of about 1-2 μL (e.g., 2 μL) of secondary antibody per 98 μL of the cell suspension.

[0046] In steps (A3) and (B3) of the method, the incubation can be an incubation at room temperature for 30 min.

[0047] In steps (A3) and (B3) of the method, after the incubation, the method can further comprise a centrifugation and a washing; the centrifugation can be a centrifugation at 600 g for 3 min; the washing can be a washing with the aforementioned NP40-Digitonin Wash Buffer.

[0048] In the detailed embodiments of the application, the centrifugation and the washing are performed sequentially and alternately, and the centrifugation and the washing are performed 3 times each.

[0049] In steps (A4) and (B4) of the method, before the addition of the ChiTag transposase, the method can further comprise a centrifugation and a resuspension of the cells; the centrifugation can be a centrifugation at 600 g for 3 min; the resuspension of the cells can be a resuspension with NP40-Dig-med-buffer (chitag enzyme incubation buffer).

[0050] The NP40-Dig-med-buffer (chitag enzyme incubation buffer) comprises the following components: 0.01% (v / v) NP40; 0.01% digitonin; 20 mM HEPES, pH 7.5; 300 mM NaCl; 0.5 mM spermidine; 1x Protease inhibitor cocktail (Sigma-Aldrich, Cat. No. 11873580001).

[0051] The resuspension of the cells can be about 50,000 cells resuspended in 99 μL of NP40-Dig-med-buffer.

[0052] In steps (A4) and (B4) of the method, PrimerA, ChIP-Tn5-PrimerB and ChIP-Tn5-PrimerC are added together with the ChiTag transposase.

[0053] The PrimerA is a single-stranded DNA shown in SEQ ID No.1 with a phosphate group modified at the 5' end; the ChIP-Tn5-PrimerB is a mixture of the four single-stranded DNAs shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5; the ChIP-Tn5-PrimerC is a mixture of the four single-stranded DNAs shown in SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9.

[0054] In steps (A4) and (B4) of the method, the ChiTag transposase may be added to the resuspended cell suspension.

[0055] The ChiTag transposase was added at a ratio of approximately 1 μL of pA-Tn5 adapter complex to 99 μL of cell suspension. The pA-Tn5 adapter complex was prepared by mixing the ChiTag transposase and the ChIP double-stranded adapter mixture at a 1:1 molar ratio and incubating at 25°C for 1 hour. The final concentration of the ChiTag transposase in the pA-Tn5 adapter complex was 5.75 pmol / μL. The ChIP double-stranded adapter mixture contained PrimerA, ChIP-Tn5-PrimerB, and ChIP-Tn5-PrimerC.

[0056] Furthermore, the ChIP double-stranded adapter mixture can be prepared according to a method comprising the following steps: Step 1, preparing the following reaction systems: Reaction System I: Primer A and ChIP-Tn5-Primer B are mixed in equal moles, specifically, Primer A (100 μM) 10 μl; ChIP-Tn5-Primer B (100 μM, final concentration of each primer is 25 μM) 10 μl. Reaction System II: Primer A and ChIP-Tn5-Primer C are mixed in equal moles, specifically, Primer A (100 μM) 10 μl; ChIP-Tn5-Primer C (100 μM, final concentration of each primer is 25 μM) 10 μl. Step 2, Reaction System I and Reaction System II are subjected to the following reaction schedule (heated lid temperature 105°C): 75°C for 15 min; 60°C for 10 min; 50°C for 10 min; 40°C for 10 min; and 25°C for 30 min. In the third step, after the reaction is completed, the reaction system I and the reaction system II are mixed in equal volumes to obtain the ChIP double-stranded adapter mixture.

[0057] In steps (A4) and (B4) of the method, the incubation may be performed at room temperature for 1 hour.

[0058] In steps (A4) and (B4) of the method, the incubation may be followed by a centrifugation and washing step; the centrifugation may be performed at 300 g for 3 minutes; and the washing may be performed using the NP40-Dig-med-buffer (chitag enzyme incubation buffer) described above.

[0059] In a specific embodiment of the present invention, the centrifugation and the washing are performed alternately in sequence, with a total of 4 centrifugations and 3 washes; the supernatant is discarded after each centrifugation.

[0060] In steps (A5) and (B5) of the method, the reaction reagents can be directly added to the cell pellet obtained after the final centrifugation in step (A4).

[0061] In steps (A5) and (B5) of the method, the reaction reagent may be Tagmentation Buffer (chitag enzyme fragmentation buffer).

[0062] The Tagmentation Buffer (chitag enzyme disruption buffer) is a solution obtained by adding 10 mM MgCl2 to the NP40-Dig-med-buffer (chitag enzyme incubation buffer) described above.

[0063] In steps (A5) and (B5) of the method, the incubation may be performed at 37° C. for 60 min.

[0064] In steps (A5) and (B5) of the method, after incubation at 37°C for 60 minutes, the mixture was centrifuged at 300 g for 3 minutes, and then conventional Tn5 transposase was added and reacted at 37°C and 500 rpm for 30 minutes.

[0065] In steps (A5) and (B5) of the method, primerA, ATAC-Tn5-primerB, and ATAC-Tn5-PrimerC are added together with the conventional Tn5 transposase;

[0066] The PrimerA is a single-stranded DNA shown in SEQ ID No. 1 with a phosphate group modified at the 5' end; the ATAC-Tn5-primerB is a mixture of the four single-stranded DNAs shown in SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, and SEQ ID No. 13; and the ATAC-Tn5-PrimerC is a mixture of the four single-stranded DNAs shown in SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, and SEQ ID No. 17.

[0067] Furthermore, the Tn5 transposase can be added in the form of an ATAC transposition mix reagent; each 25 μl of the ATAC transposition mix reagent contains 5 μl of 5X TAG Buffer (BGE005B01), 16 μl of 1% BSA / PBS (A0332), and 4 μl of Tn5 adaptor complex; the Tn5 adaptor complex is obtained by uniformly mixing Tn5 transposase and a Tn5 double-stranded adapter mixture in a molar ratio of 1:1, and incubating at 25°C for 1 hour, wherein the final concentration of Tn5 transposase in the Tn5 adaptor complex is 0.875 U / μl; the Tn5 double-stranded adapter mixture contains PrimerA, the ATAC-Tn5-primerB, and the ATAC-Tn5-PrimerC.

[0068] Furthermore, the Tn5 double-stranded adapter mixture can be prepared according to a method comprising the following steps: Step 1, preparing the following reaction system: Reaction system I: the Primer A and the ATAC-Tn5-primer B are mixed in equal moles, specifically, Primer A (100 μM) 10 μl; ATAC-Tn5-primer B (100 μM, the final concentration of each primer is 25 μM) 10 μl; Reaction system II: the Primer A and the ATAC-Tn5-Primer C are mixed in equal moles, specifically, Primer A (100 μM) 10 μl; ATAC-Tn5-Primer C (100 μM, final concentration of each primer is 25 μM) 10 μl; second, reaction system I and reaction system II are subjected to the following reaction program (heated lid temperature 105°C): 75°C 15 min; 60°C 10 min; 50°C 10 min; 40°C 10 min; 25°C 30 min; third, after the reaction is completed, reaction system I and reaction system II are mixed with equal volumes to obtain the Tn5 double-stranded adapter mixture.

[0069] The addition ratio of the reaction reagent (Tagmentation Buffer) and the ATAC transposition mixed reagent can be approximately: 300 μL of the reaction reagent (Tagmentation Buffer) and / or 25 μL of the ATAC transposition mixed reagent per 50,000 cells.

[0070] In step (A6) of the method, the single-cell droplets are generated by utilizing the oil-in-water structure, specifically by utilizing the BGI DNBelab C4 portable single-cell system.

[0071] In step (A6) of the method, the primers used for the in-droplet amplification are: Tn-Primer and 183+C Primer. The Tn-Primer is a single-stranded DNA shown in SEQ ID No. 18, and the 183+C Primer is a single-stranded DNA shown in SEQ ID No. 19.

[0072] In a specific embodiment of the present invention, the reaction procedure for the amplification is: 72°C for 30 min; 98°C for 30 s; 98°C for 10 s, 63°C for 30 s, 72°C for 1 min, 10 cycles; 72°C for 5 min; and insulation at 4°C.

[0073] In step (A7) of the method, the demulsification can be achieved by the following steps: transferring the droplets amplified in (A6) into a low adsorption centrifuge tube, adding Additive B (Perfluoro-1-octnaol, A63881), inverting to mix, centrifuging at 1000g for 1 min, and then placing on a magnetic stand for 1 min, removing the liquid.

[0074] Further, the method can further comprise the following steps: adding Wash Buffer F (10 ml TE Buffer, 10 μl 10% Tween-20, AM9820), inverting to mix, centrifuging at 1000g for 1 min, and then placing on a magnetic stand for 1 min, removing the supernatant. This step can be repeated once. After removing the supernatant, ATAC Enzyme II treatment is performed, and the mixture is incubated in a metal bath at 37°C at 1000 rpm for 45 min.

[0075] Further, after the enzyme treatment is completed, the method can further comprise the following steps: briefly centrifuging, adding Wash Buffer E (9.75 ml TE Buffer, 0.25 ml 20% SDS, AM9820), inverting to mix, and terminating the reaction; centrifuging at 1000g for 1 min, and then placing on a magnetic stand for 1 min, removing the liquid.

[0076] Further, the method can further comprise the following steps: adding Wash Buffer F, inverting to mix, centrifuging at 1000g for 1 min, and then placing on a magnetic stand for 1 min, removing the supernatant. This step can be repeated twice.

[0077] In step (A7) of the method, the primers used in the amplification are Tn-Primer and 183-pho Primer; the Tn-Primer is a single-stranded DNA as shown in SEQ ID No. 18, and the 183-pho Primer is a single-stranded DNA as shown in SEQ ID No. 20 modified with a phosphate group at the 5' end.

[0078] Furthermore, the amplification can be specifically performed according to a method comprising the following steps: after washing the magnetic beads and removing the supernatant, PCR Ready Mix (containing ATAC Enzyme III (2×KAPA HiFi HotStart Ready Mix, KK2602), Tn-Primer, 183-pho Primer, 60% Optiprep Density Gradient Medium (D1556-250ML), NF-H2O) was added, the magnetic beads were pipetted to mix and the mixture was evenly divided into eight tubes, the centrifuge tube was washed again with PCR Ready Mix, and the mixture was evenly divided into eight tubes (operated at 4°C to avoid inactivation of PCR Ready Mix).

[0079] In a specific embodiment of the present invention, the reaction procedure for the amplification is: 98°C for 30s; 98°C for 10s, 63°C for 30s, 72°C for 1min, 15-20 cycles (such as 15 cycles); 72°C for 5min; and insulation at 4°C.

[0080] In step (A7) of the method, the purification may be performed by adding 1.2 times the volume of magnetic beads to the amplified product for purification (ie, 1.2× magnetic bead purification).

[0081] In step (A7) of the method, the magnetic beads may be Agencourt AMPure XP magnetic beads.

[0082] In step (B6) of the method, the primers used for the amplification are Bulk-N5 Primer and 183+C-pho Primer; the Bulk-N5 Primer is a single-stranded DNA shown in SEQ ID No. 22; the 183+C-phoPrimer is a single-stranded DNA shown in SEQ ID No. 23 with a phosphate group modified at the 5' end.

[0083] Furthermore, the reaction procedure for the amplification is: 72°C for 5 min; 98°C for 30 s; 98°C for 10 s, 63°C for 30 s, 72°C for 5 s, 15 cycles; 72°C for 1 min; and insulation at 12°C.

[0084] In steps (A8) and (B7) of the method, a cyclization step may be included before the enzymatic cleavage. Furthermore, the cyclization may be achieved with the aid of 153+181 Splint oligo.

[0085] The 153+181 splint oligo is a single-stranded DNA shown in SEQ ID No. 21.

[0086] Furthermore, a single-strand heat denaturation reaction is performed first, and then a cyclization reaction is performed.

[0087] The 153+181 Splint oligo was added to the reaction system of the single-strand thermal denaturation reaction.

[0088] In a specific embodiment of the present invention, the reaction system for single-strand thermal denaturation is: 200-400 ng of purified DNA mixture, 3 μL of the 153+181 Splint oligo with a concentration of 20 μM; and NF-H2O to make up to 50 μL.

[0089] In a specific embodiment of the present invention, the reaction conditions for the single-strand thermal denaturation are: 95° C. for 3 min; and insulation at 4° C.

[0090] In a specific embodiment of the present invention, the reaction system of the cyclization reaction is: 50 μL of the above-mentioned single-stranded heat denaturation reaction product; 6 μL of 10×TA buffer (EPICENTRE TA6160); 0.6 μL of 100 mM ATP; 0.6 μL of 400 U / μL T4 DNA ligase; and 2.8 μL of TE Buffer.

[0091] In a specific embodiment of the present invention, the reaction conditions of the cyclization reaction are: 37° C. for 45 min; and insulation at 4° C.

[0092] In steps (A8) and (B7) of the method, the enzymes used for the enzymatic cleavage may be EXO I enzyme (NEBM0293L) and EXO III enzyme (NEB M0206L).

[0093] In a specific embodiment of the present invention, the enzyme digestion reaction system is: 60 μL of the ligation product obtained from the above cyclization reaction; 0.4 μL of 10×TA buffer (EPICENTRE TA6160); 1.95 μL of EXO I enzyme with a concentration of 20 U / μL; 0.65 μL of EXO III enzyme with a concentration of 100 U / μL; and 1 μL of TE Buffer.

[0094] In a specific embodiment of the present invention, the reaction conditions of the enzyme digestion are: 37° C. for 30 min and incubation at 4° C. After the reaction is completed, 4 μl of 0.1 mM EDTA (AMBION AM9260G) is added to terminate the reaction.

[0095] In steps (A8) and (B7) of the method, the step of purifying the cleavage products using magnetic beads may be further included after the enzymatic cleavage.

[0096] In steps (A8) and (B7) of the method, the magnetic beads used to purify the enzyme-cleaved products may be PEG32 beads.

[0097] Furthermore, 90 μL of PEG32 beads were added to 64 μL of the above-mentioned enzyme digestion product, and then the following operations were performed: vortex shaking to mix, incubate at room temperature for 10 minutes; place in a magnetic stand to separate the magnetic beads and liquid; remove the supernatant after the solution is clarified; keep the reaction tube in the magnetic stand, add 80% ethanol to rinse the magnetic beads; incubate at room temperature for 30 seconds and remove the supernatant; keep the reaction tube in the magnetic stand, open the lid and air dry the magnetic beads for 3 minutes; remove the reaction tube from the magnetic stand, add sterile ultrapure water to elute; mix; place the reaction tube in a magnetic stand to separate the magnetic beads and liquid; after the solution is clarified, aspirate the supernatant into a sterilized tube and store at -20°C (the result here is the final library).

[0098] In steps (A9) and (B8) of the method, the sequencing is high-throughput sequencing, specifically paired-end sequencing, and the sequencing method can be specifically PE50+26+10 (paired-end sequencing, measuring 50bp at each end, barcode1 sequence measuring 26bp, and barcode2 sequence measuring 10bp).

[0099] In the method, the test cells can specifically be tumor cells K562.

[0100] In a second aspect, the present invention claims a method for constructing a DNA library for multi-dimensional analysis of cellular epigenomics.

[0101] The method for constructing a DNA library for multidimensional analysis of single-cell epigenomics claimed in the present invention may specifically include steps (A1)-(A8) of method A described in the first aspect above or steps (B1)-(B7) of method B.

[0102] In a third aspect, the present invention claims a kit.

[0103] The kit claimed in the present invention may contain ChiTag transposase, conventional Tn5 transposase, and other related reagents of CUT&Tag technology and / or ATAC-seq technology. The kit is used for:

[0104] (B1) Multidimensional analysis of cellular epigenomics; or

[0105] (B2) Construction of DNA libraries for multidimensional analysis of cellular epigenomics.

[0106] Furthermore, other related reagents of the CUT&Tag technology and / or ATAC-seq technology can be selected from all or part of the following: the NP40-digoxigenin wash buffer added with 1.5-2.5 mM (such as 2 mM) EDTA as described above; the NP40-digoxigenin wash buffer (NP40-Digitonin Wash Buffer) described above; the basic wash buffer (Wash Buffer) described above; the antibody corresponding to the specific protein described above; the secondary antibody described above; the NP40-Dig-med-buffer (chitag enzyme incubation buffer) described above; the Tagmentation Buffer (chitag enzyme shearing buffer) described above; the termination reaction solution (4×Stop Buffer) described above; the PrimerA (modified with a phosphate group at the 5' end, SEQ ID No. 1) described above; the ATAC-Tn5-PrimerB (SEQ ID No. 10-13) described above; the ATAC-Tn5-PrimerC (SEQ ID No.14-17); ChIP-Tn5-PrimerB described above (SEQ ID No.2-5); ChIP-Tn5-PrimerC described above (SEQ ID No.6-9); Tn-Primer described above (SEQ ID No.18); 183+C Primer described above (SEQ ID No.19); 183-pho Primer described above (5' end modified with a phosphate group, SEQ ID No.20); AgencourtAMPure XP magnetic beads; Bulk-N5 Primer described above (SEQ ID No.22); 183+C-pho Primer described above (SEQ ID No.23); 153+181 Splint oligo described above (SEQ ID No.21); EXO I enzyme; EXO III enzyme; PEG32 beads.

[0107] Furthermore, the kit may also contain a readable carrier (such as paper, or a data storage device such as a CD, USB flash drive, etc.) recording the method described in the first or second aspect above.

[0108] In a fourth aspect, the present invention claims a system.

[0109] The system claimed in the present invention comprises the aforementioned kit, as well as instruments and equipment related to CUT&Tag technology and / or ATAC-seq technology. The system is used to:

[0110] (B1) multi-dimensionally resolving cell epigenomics; or

[0111] (B2) constructing DNA library for multi-dimensionally resolving cell epigenomics.

[0112] Further, the CUT&Tag technology and / or ATAC-seq technology related instrument equipment can be selected from all or part of the following: high-throughput sequencer (such as BGISEQ500 sequencer); PCR instrument; DNBelab C4 portable single-cell system; centrifuge; shaker; microscope; cell counting plate.

[0113] In a fifth aspect, the present application claims the use of the kit or the system as described above in any one of the following:

[0114] (B1) multi-dimensionally resolving cell epigenomics;

[0115] (B2) constructing DNA library for multi-dimensionally resolving cell epigenomics.

[0116] In a sixth aspect, the present application claims the use of the method or the kit or the system as described above in any one of the following:

[0117] (C1) studying cell population heterogeneity related to development and / or disease;

[0118] (C2) mapping cell atlas;

[0119] (C3) resolving tumor cells with different clinical characteristics;

[0120] (C4) clinically studying tumor cell evolution and / or metastasis.

[0121] In the present application, the multi-dimensionally resolving cell epigenomics can be multi-dimensionally resolving single cell epigenomics or multi-dimensionally resolving multi-cell epigenomics. BRIEF DESCRIPTION OF DRAWINGS

[0122] Figure 1 A schematic diagram of the CUT&Tag method.

[0123] Figure 2 A schematic diagram of the ATAC-seq method. A is a schematic diagram of Tn5 transposase binding to chromatin open region to break genomic sequence; B is a schematic diagram of ATAC-seq library preparation structure.

[0124] Figure 3 A quality inspection result diagram of DNA product obtained from single cell.

[0125] Figure 4 A quality inspection result diagram of DNA product obtained from multi-cell.

[0126] Figure 5 Unique reads number for single-cell CUT&Tag data.

[0127] Figure 6 Unique reads number for single-cell ATAC-seq data.

[0128] Figure 7 Fragment distribution for single-cell omics CUT&Tag.

[0129] Figure 8 Fragment distribution for single-cell omics ATAC-seq.

[0130] Figure 9 Fragment distribution for multi-cell omics CUT&Tag.

[0131] Figure 10 Fragment distribution for multi-cell omics ATAC-seq.

[0132] Figure 11 Peak distribution plot for CUT&Tag partial data.

[0133] Figure 12 Peak distribution plot for ATAC-seq partial data.

[0134] Best mode of carrying out the invention

[0135] The following examples facilitate a better understanding of the present application, but do not limit the present application. The experimental methods in the following examples are all conventional methods, unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent stores, unless otherwise specified. The quantitative experiments in the following examples are all set up with at least three repeated experiments, and the results are averaged.

[0136] Example 1, Multi-dimensional resolution of cellular epigenomics

[0137] I. Multi-dimensional resolution of single-cell epigenomics

[0138] Test cell: K562 cell.

[0139] 1. Cell pretreatment (0.5-1 h)

[0140] Note: All steps before cell permeation are performed at room temperature to minimize stress on cells. Avoid cavitation and vigorous vortexing during resuspension.

[0141] 1.1 Collect fresh cells at room temperature and count (500,000-1,000,000 cells). Centrifuge at low speed (600 × g for 3 min) at room temperature (23-28°C, the same below) and remove the liquid.

[0142] 1.2 Add at least 1 volume of Wash Buffer to resuspend the cells, centrifuge at low speed (600 × g for 3 min) at room temperature, and aspirate the liquid;

[0143] Wash Buffer: 20mM HEPES, pH 7.5; 150mM NaCl; 0.5mM Spermidine (spermidine); 1×Protease inhibitor cocktail (Sigma-Aldrich, Cat. No. 11873580001).

[0144] Note: The initial wash and incubation steps were centrifuged at 600 g for 3 min, and after binding of pA-Tn5, the cells were centrifuged at 300 g for 3 min.

[0145] 2. Change cell permeability

[0146] The cell nuclei were isolated by changing the cell permeability with NP40-Digitonin Wash Buffer, and the cells were resuspended in 1 ml of NP40-Digitonin Wash Buffer.

[0147] NP40-Digitonin Wash Buffer: A solution prepared by adding 0.01% (volume percentage) NP40 and 0.01% digoxin to wash buffer. Digoxin is BN2006# Digitonin (5%), Invitrogen brand, size 1, with a weight percentage of 5. The digoxin concentration in the NP40-Digitonin Wash Buffer is equivalent to the digoxin concentration of the commercial product after a 500-fold dilution. References to 0.01% digoxin below shall have the same meaning.

[0148] Centrifuge at 600 g for 3 min, discard the supernatant, and resuspend in 49 μl NP40-Digitonin Wash Buffer supplemented with 2 mM EDTA.

[0149] 3. Binding primary antibody (CUT&Tag)

[0150] 3.1 Add 1 μl of primary antibody (H3K27me3 antibody, Cell Signaling Technology, 9733, Lot 14) to the above sample at a volume ratio of 1:50 and gently vortex (the default volume ratio is 1:50-1:100 or the immunoblot concentration recommended in the instructions).

[0151] 3.2 Place all sample tubes on a shaker and incubate at room temperature for 2 hours (or overnight at 4°C). While shaking, the liquid should remain at the bottom and sides of the tubes.

[0152] 4. Binding secondary antibody

[0153] 4.1 Remove all sample tubes from the shaker, add 1 ml of NP40-Digitonin Wash buffer to rinse once, centrifuge at 600g for 3 minutes, aspirate the supernatant, and add 98 μl of NP40-Digitonin Wash buffer to resuspend the cells;

[0154] 4.3 Add 2 μl of secondary antibody (Guinea Pig anti-Rabbit IgG (Heavy & Light Chain) antibody (Antibodies-Online ABIN101961)) to 98 μl of NP40-Digitonin Wash buffer at a volume ratio of 1:50 to each tube of sample and gently vortex to mix the liquid evenly;

[0155] 4.4 Place all sample tubes on a shaker and incubate at room temperature for 30 minutes;

[0156] 4.5 Remove all sample tubes from the shaker, centrifuge at 600g for 3 minutes to precipitate, and aspirate the supernatant;

[0157] 4.6 Add 1 ml of NP40-Digitonin Wash buffer to each tube of sample and invert 10 times or gently vortex to mix the liquid and remove unbound antibodies;

[0158] 4.7 Repeat steps 4.5 and 4.6 twice.

[0159] 5. Combining ChiTag transposomes

[0160] 5.1 Centrifuge all sample tubes at 600g for 3 minutes and remove the supernatant;

[0161] 5.2 Add 100 μl NP40-Dig-med-buffer to resuspend the cells and count them. Based on the count results, take 50,000 cells and resuspend them in NP40-Dig-med-buffer to 99 μl. Add 1 μl of pA-Tn5 adapter complex.

[0162] Among them, ChiTag transposase and ChIP double-stranded adapter mixture were mixed evenly at a molar ratio of 1:1 and incubated at 25°C for 1 hour to obtain the pA-Tn5 adaptor complex. The reaction system example is shown in Table 1.

[0163] Table 1 Examples of pA-Tn5 adaptor complex reaction systems

[0164] ChIP double linker mix (50 pmol / μl) 1 μl ChiTag™ (6.5 pmol / μl) 7.7 μl

[0165] Note: The manufacturer's product number of ChiTag transposase is Novoprotein M058-YH01.

[0166] The ChIP double-stranded adapter mixture was prepared as follows:

[0167] a. Dissolve Primer A, ChIP-Tn5-Primer B, and ChIP-Tn5-Primer C to 100 μM in Annealing Buffer (formula: 100 μl 1 M Tris-HCl, pH 7.8; 20 μl 0.5 M EDTA, pH 8.0; 100 μl 5 M NaCl; 9.78 ml NF-H2O).

[0168] PrimerA: 5'-Pho-CTGTCTCTTATACACATCT-3' (SEQ ID No. 1);

[0169] ChIP-Tn5-PrimerB: (4 primers diluted to 100 μM and mixed in equal volumes)

[0170] 5'-TCGTCGGCAGCGTCAGGCGAAGGCGATCGAGGACCGGCAGATGTGTATAAGAGACAG-3' (SEQ ID No. 2);

[0171] 5'-TCGTCGGCAGCGTCTAATCTTAGCGATCGAGGACCGGCAGATGTGTATAAGAGACAG-3' (SEQ ID No. 3);

[0172] 5'-TCGTCGGCAGCGTCCAGGACGTGCGATCGAGGACCGGCAGATGTGTATAAGAGACAG-3' (SEQ ID No. 4);

[0173] 5'-TCGTCGGCAGCGTCGTACTGACGCGATCGAGGACGGCAGATGTGTATAAGAGACAG-3' (SEQ ID No. 5).

[0174] ChIP-Tn5-PrimerC: (4 primers diluted to 100 μM and mixed in equal volumes)

[0175] 5'-GTCTCGTGGGCTCGGAATCTATCAACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG-3' (SEQID No. 6);

[0176] 5'-GTCTCGTGGGCTCGGACCAGGAAGGCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG-3' (SEQID No. 7);

[0177] 5'-GTCTCGTGGGCTCGGGAGAGATATTCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG-3' (SEQID No. 8);

[0178] 5'-GTCTCGTGGGCTCGGGGGAAACATGCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG-3' (SEQ ID No. 9).

[0179] b. Prepare the following reaction systems respectively:

[0180] Reaction system I: Primer A (100 μM) 10 μl; ChIP-Tn5-Primer B (100 μM) 10 μl.

[0181] Reaction system II: Primer A (100 μM) 10 μl; ChIP-Tn5-Primer C (100 μM) 10 μl.

[0182] c. Vortex and thoroughly mix Reaction System I and Reaction System II, then briefly centrifuge to allow the solution to return to the bottom of the tube. Place in a PCR instrument and perform the following reaction program (heated lid temperature 105°C): 75°C for 15 minutes; 60°C for 10 minutes; 50°C for 10 minutes; 40°C for 10 minutes; and 25°C for 30 minutes.

[0183] d. After the reaction is completed, equal volumes of reaction system I and reaction system II are mixed and mixed to obtain the ChIP double-stranded adapter mixture, which is then stored at -20°C.

[0184] 5.3 Place all sample tubes on a shaker and incubate at room temperature for 1 hour;

[0185] 5.4 Remove all sample tubes from the shaker, centrifuge at 300g for 3 minutes, and remove the supernatant;

[0186] 5.5 Add NP40-Dig-med-buffer to each tube of sample and invert 10 times or gently vortex to mix the solution evenly;

[0187] 5.6 Repeat steps 5.4 and 5.5 twice, then centrifuge at 300g for 3 minutes. Aspirate the supernatant to remove excess unbound pA-Tn5 adapter complex.

[0188] NP40-Dig-med-buffer: 0.01% (volume percentage) NP40; 0.01% digoxin; 20 mM HEPES, pH 7.5; 300 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail (Sigma-Aldrich, Cat. No. 11873580001).

[0189] 6. Fragmentation (1h)

[0190] 6.1 Add 300 μl Tagmentation Buffer to each tube of sample and vortex gently while adding;

[0191] 6.2 Incubate at 37°C and 200 rpm for 60 min, then centrifuge at 300 g for 3 min and remove the supernatant.

[0192] 6.3 Add 25 μl of ATAC transposition mix (5 μl of 5X TAG Buffer (BGE005B01), 16 μl of 1% BSA / PBS (A0332), 4 μl of Tn5 adaptor complex) to the sample tube and incubate at 37°C, 500 rpm, for 30 min.

[0193] Among them, Tn5 transposase and Tn5 double-stranded adapter mixture were evenly mixed at a molar ratio of 1:1, and incubated at 25°C for 1 hour to obtain Tn5 adaptor complex. The reaction system example is shown in Table 2.

[0194] Table 2 Examples of Tn5 adaptor complex reaction systems

[0195] Tn5 double linker mix (50 pmol / μl) 1 μl Tn5 transposase (0.875 U / μl, self-made, Cat. No. BGE005) 7.7 μl

[0196] The Tn5 enzyme double-stranded adapter mixture is prepared as follows:

[0197] a. Dissolve Primer A, ATAC-Tn5-Primer B, and ATAC-Tn5-Primer C to 100 μM in Annealing Buffer (formula: 100 μl 1 M Tris-HCl, pH 7.8; 20 μl 0.5 M EDTA, pH 8.0; 100 μl 5 M NaCl; 9.78 ml NF-H2O).

[0198] PrimerA: 5'-Pho-CTGTCTCTTATACACATCT-3' (SEQ ID No. 1);

[0199] ATAC-Tn5-Primer B: (4 primers diluted to 100 μM and mixed in equal volumes)

[0200] 5'-TCGTCGGCAGCGTCTATAGCCTGCGATCGAGGACCGGCAGATGTGTATAAGAGACAG-3' (SEQ ID No. 10);

[0201] 5'-TCGTCGGCAGCGTCATAGAGGCGCGATCGAGGACGGCAGATGTGTATAAGAGACAG-3' (SEQ ID No. 11);

[0202] 5'-TCGTCGGCAGCGTCCCTATCCTGCGATCGAGGACCGGCAGATGTGTATAAGAGACAG-3' (SEQ ID No. 12);

[0203] 5'-TCGTCGGCAGCGTCGGCTCTGAGCGATCGAGGACCGGCAGATGTGTATAAGAGACAG-3' (SEQ ID No. 13).

[0204] ATAC-Tn5-Primer C: (4 primers diluted to 100 μM and mixed in equal volumes)

[0205] 5'-GTCTCGTGGGCTCGGATTTATGACACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG- 3' (SEQ ID No. 14);

[0206] 5'-GTCTCGTGGGCTCGGCCTTAATTAACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG-3' (SEQ ID No. 15);

[0207] 5'-GTCTCGTGGGCTCGGTCAGTGAGTCCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG-3' (SEQ ID No. 16);

[0208] 5'-GTCTCGTGGGCTCGGACTGCCTTATCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG-3' (SEQ ID No. 17).

[0209] b. Prepare the following reaction systems respectively:

[0210] Reaction system I: Primer A (100 μM) 10 μl; ATAC-Tn5-Primer B (100 μM) 10 μl.

[0211] Reaction system II: Primer A (100 μM) 10 μl; ATAC-Tn5-Primer C (100 μM) 10 μl.

[0212] c. Vortex and thoroughly mix Reaction System I and Reaction System II, then briefly centrifuge to allow the solution to return to the bottom of the tube. Place in a PCR instrument and perform the following reaction program (heated lid temperature 105°C): 75°C for 15 minutes; 60°C for 10 minutes; 50°C for 10 minutes; 40°C for 10 minutes; and 25°C for 30 minutes.

[0213] d. After the reaction is completed, equal volumes of reaction system I and reaction system II are mixed and mixed to obtain the Tn5 double-stranded adapter mixture, which is then stored at -20°C.

[0214] 7. Droplet Generation & PCR

[0215] 7.1 Magnetic Bead Preparation

[0216] 7.1.1 Magnetic beads (Spherotech, USA, catalog number SVM-200-4, https: / / www.spherotech.com / coa_mag_par.htm) were coated with oligonucleotides on their surfaces, using the method described in the stLFR magnetic bead preparation article published by BGI (Efficient and unique co-barcoding of second-generation sequencing reads from long DNA molecules enabling cost-effective and accurate sequencing, haplotyping, and de novoassembly, https: / / genome.cshlp.org / content / early / 2019 / 04 / 02 / gr.245126.118) to obtain surface-coated oligonucleotide-coated magnetic beads (Tn-Beads).

[0217] 7.1.2 Pipette 300,000 Tn-Beads into a 0.2 ml low-adsorption PCR tube, place on a magnetic rack for 2 minutes, and remove the supernatant.

[0218] 7.1.3 Remove the PCR tube from the magnetic stand and add 200 μl of 1× Wash Buffer A (1 mM EDTA, AM9260, 9 mg / ml 85% KOH, P5958-250G). Mix the beads by pipetting using a 200 μl low-retention pipette tip. Incubate at room temperature for 5 min.

[0219] 7.1.4 After incubation, place the PCR tube on a magnetic rack for 2 minutes and remove the supernatant.

[0220] 7.1.5 Remove the PCR tube from the magnetic rack and add 200 μl of 1× Wash Buffer A. Use a 200 μl low-absorption pipette tip to mix the magnetic beads. Place the PCR tube on the magnetic rack and let it stand for 2 minutes. Remove the supernatant.

[0221] 7.1.6 Remove the PCR tube from the magnetic rack and add 200 μl Wash Buffer B (500 μl 1M Tris-HCl, 15567027, 300 μl 5M NaCl, S5150, 50 μl 10% Tween-20, 9.15 ml H2O). Use a 200 μl low-absorption pipette tip to mix the magnetic beads. Place the PCR tube on the magnetic rack and let it stand for 2 minutes. Remove the supernatant and repeat the above steps once.

[0222] 7.1.7 Add 200 μl Wash Buffer B. Once the nuclei are prepared, remove the supernatant as much as possible.

[0223] 7.1.8 Remove the PCR tube from the magnetic stand, add 100 μl of Beads Resuspension Buffer, resuspend the magnetic beads, and place on ice until ready for loading.

[0224] 7.1.9 Prepare Beads Resuspension Buffer according to Table 2 below:

[0225] Table 2 Beads Resuspension Buffer preparation system

[0226] Composition Amount (μl) 0.1% SDS 40 ATAC Bead Buffer 58 Tn Primer (20 μM) 1 183+C Primer (20 μM) 1 Total volume 100

[0227] ATAC Bead Buffer: 20μl 5x Fidelity Buffer (KK2102), 3μl 10mM dNTP Mix (18427013), 7μl 25mM MgCl2 (20303), 16.7ul 60% Optiprep Density Gradient Medium (D1556-250ML), 11.3μl NF-H2O.

[0228] Tn Primer: 5'-CGTAGCCATGTCGTTCTG-3' (SEQ ID No. 18);

[0229] 183+C Primer: 5'-GAGACGTTCTCGACTCAGCAGAGTCTCGTGGGCTCGG-3' (SEQ ID No. 19).

[0230] 7.2 Nucleus Preparation

[0231] 7.2.1 Count the nuclei of cells transposed in step 6.3. Based on the nuclei concentration, aspirate 10,000 nuclei and add them to the Nuclei Resuspension Buffer.

[0232] 7.2.2 Prepare Nuclei Resuspension Buffer according to Table 3 below

[0233] Table 3 Nuclei Resuspension Buffer preparation system

[0234] Composition Amount (μl) Nuclei n KAPA Hifi DNA polymerase (KK2102) 8 ATAC Nuclei Buffer 46.7 <![CDATA[NF-H2O]]> 45.3-n Total volume 100

[0235] ATAC Nuclei Buffer: 20μl 5x Fidelity Buffer (KK2102), 3μl 10mM dNTP Mix (18427013), 7μl 25mM MgCl2 (20303), 16.7μl 60% Optiprep Density Gradient Medium (D1556-250ML).

[0236] 7.3 Droplet Generation (Using the BGI DNBelab C4 Portable Single-Cell System to Generate Droplets)

[0237] 7.3.1 Remove the protective film from the chip (Dow Corning 184) and place it in the chip slot area of ​​the droplet generator.

[0238] 7.3.2 Add 100 μl of Bio-rad Oil (1864006) to the collection tube, tighten the collection cap, and place the collection tube upright on a fixed stand;

[0239] 7.3.3 Insert end A of the connecting tube on the lid (the connecting tube that contacts the bottom of the collection tube) into the outlet hole of the chip;

[0240] 7.3.4 Adjust the initial position of the 50ml syringe piston to the 28ml mark and place it on the fixed stand. Use a needle to connect the syringe to the connecting tube end B on the collection tube cap (not the connecting tube at the bottom of the collection tube);

[0241] 7.3.5 Use a pipette to gently pipette and mix the cells. Add 100 μl of cell suspension to the cells well of the chip, ensuring that no bubbles appear at the bottom of the well.

[0242] 7.3.6 Gently pipette to mix the beads and add 100 μl of beads to the beads well of the chip, ensuring that no bubbles appear at the bottom of the well.

[0243] 7.3.7 Quickly add Bio-rad Oil (add 400 μl initially, as oil will need to be added during the experiment) to the Oil well of the chip;

[0244] 7.3.8 Quickly pull the syringe piston to the 30ml mark and secure the syringe to the mounting bracket;

[0245] 7.3.9 Start the timer, collect the droplets (you can wait until the liquid runs out during the test phase), and make sure to add oil;

[0246] 7.3.10 After droplet generation is complete, immediately loosen the collection cap on the collection tube, remove the connecting tube from the chip outlet hole, stretch the connecting tube vertically to allow the droplets in the tube to flow into the collection tube, and then replace the collection tube cap with a standard one.

[0247] 7.3.11 Transfer droplet to octet tube, note that the droplet level does not exceed 100 μl, then add 100 μl mineral oil on top of the droplet, cover the octet tube cap and perform PCR according to Table 4 below:

[0248] Table 4 PCR program in droplet

[0249]

[0250] Stop point: After PCR is finished, the droplet can be left at 4 °C for 72 h.

[0251] 8. Emulsification & amplification outside droplet

[0252] 8.1 Emulsification

[0253] 8.1.1 After PCR is finished, transfer the droplet to a new low-retention 1.5 ml centrifuge tube, add 100 μl Additive B (Perfluoro-1-octnaol, A63881), mix by inversion, centrifuge at 1000 g for 1 min, then place on a magnetic stand and let stand for 1 min, remove the liquid;

[0254] 8.1.2 Add 500 μl Wash Buffer F (10 ml TE Buffer, 10 μl 10% Tween-20, AM9820), mix by inversion, centrifuge at 1000 g for 1 min, then place on a magnetic stand and let stand for 1 min, remove the supernatant;

[0255] 8.1.3 Repeat the above step once, after removing the supernatant, add the following enzyme treatment reaction system.

[0256] 8.2 Enzyme treatment

[0257] 8.2.1 Prepare the enzyme treatment reaction system in advance according to Table 5 below:

[0258] Table 5 Enzyme treatment reaction system

[0259] Composition Amount (μl) <![CDATA[NF-H2O]]> 170 ATAC Enzyme II 10 ATAC Reaction Buffer 20 Total volume 200

[0260] ATAC Enzyme II: EXO I (M0293S)

[0261] ATAC Reaction Buffer: 10x EXO I Buffer (B0293s)

[0262] 8.2.1 Take 200 μl of the reaction system and add it to the PCR tube containing the magnetic beads (no need to blow);

[0263] 8.2.3 Incubate at 37 °C in a metal bath, 1000 rpm for 45 min;

[0264] 8.2.4 After the reaction is complete, centrifuge briefly, add 1 ml of Wash Buffer E (formula: 9.75 ml TE Buffer, 0.25 ml 20% SDS, AM9820), invert and mix to terminate the reaction;

[0265] 8.2.5 Centrifuge at 1000g for 1 minute, place on a magnetic rack, let stand for 1 minute, and remove the liquid.

[0266] 8.2.6 Add 500 μl Wash Buffer F (formula: 10 ml TE Buffer, 10 μl 10% Tween-20, AM9820), mix by inversion, centrifuge at 1000 g for 1 min, place on a magnetic rack, let stand for 1 min, remove the liquid, and repeat the above steps twice;

[0267] 8.2.7 Keeping the magnetic beads adsorbed, add 400 μl of PCR Ready Mix, pipette the magnetic beads to mix evenly, and divide the mixture evenly into eight tube strips. Rinse the centrifuge tubes again with 400 μl of PCR Ready Mix, and divide the mixture evenly into eight tube strips. (Note: This step should be performed at 4°C to avoid inactivation of the PCR Ready Mix.)

[0268] 8.3 PCR amplification

[0269] 8.3.1 Prepare PCR Ready Mix according to Table 6 below:

[0270] Table 6 PCR Ready Mix preparation system

[0271] Composition Amount (μl) ATAC Enzyme III (2X KAPA HiFi HotStart Ready Mix, KK2602) 50 Tn Primer (20 μM) 2 183-pho Primer (20 μM) 2 60% Optiprep Density Gradient Medium 16.7 <![CDATA[NF-H2O]]> 29.3 Total volume 100

[0272] Tn Primer: 5'-CGTAGCCATGTCGTTCTG-3' (SEQ ID No. 18);

[0273] 183-pho Primer: 5'-pho GAGACGTTCTCGACTCAGCAGA-3' (SEQ ID No. 20).

[0274] 8.3.2 Complete the aliquoting according to 8.2.7, Vortex mix the reaction suspension, and centrifuge briefly;

[0275] 8.3.3 Perform the reaction on a PCR instrument according to the conditions in Table 7 below, with the heated lid set to 105°C.

[0276] Table 7 PCR program

[0277]

[0278] Note: The number of PCR cycles can be changed accordingly for different samples;

[0279] Stopping point: PCR products can be stored at 4°C for 24 hours. After PCR, use Qubit quantification to determine if there are any problems with the previous treatment. If the concentration is <2 ng / μl, it indicates that the sample treatment has failed and no further steps are required.

[0280] 9. 1.2× magnetic bead purification

[0281] 9.1 Take out AgencourtAMPure XP in advance and equilibrate it at room temperature for at least 30 minutes. Vortex and mix thoroughly before use.

[0282] Note: Liquid evaporation may occur during the PCR reaction. If the liquid evaporates significantly, the liquid volume V needs to be re-quantified and replenished with H2O to 800μl.

[0283] 9.2 Transfer the PCR product to a new 2 ml centrifuge tube. Pipette 960 μl of AgencourtAMPure XP into the tube, vortex until completely mixed, and incubate at room temperature for 8 minutes.

[0284] 9.3 Place on a magnetic rack and let it stand for 5 minutes until the beads are adsorbed and the liquid becomes clear.

[0285] 9.4 Discard the supernatant and avoid aspirating the magnetic beads;

[0286] 9.5 Add 200 μl of 80% ethanol, let stand for 30 seconds, discard the supernatant, and repeat this step twice;

[0287] 9.6 Place the sample on a magnetic rack and let it dry until the surface of the magnetic beads is no longer reflective (approximately 2-5 minutes).

[0288] 9.7 Remove the PCR tube from the magnetic rack, add 50-100 μl NF-H2O to dissolve, pipette 10 times to mix, and let stand at room temperature for 5 minutes;

[0289] 9.8 Place on the magnetic rack and let stand for 3 minutes until the liquid becomes clear;

[0290] 9.9 Recover the supernatant to avoid attracting the magnetic beads;

[0291] 9.10 Take 1 μl of the sample to measure the qubit concentration; dilute the product to about 2 ng / μl according to the concentration, and then take 1 μl of the sample for 2100HS detection. The library fragment length is 200 bp-600 bp.

[0292] 10. Single-strand thermal denaturation reaction (200-400ng DNA per reaction)

[0293] The reaction system is shown in Table 8, and the parameter settings are shown in Table 9.

[0294] Table 8 Reaction system for single-strand thermal denaturation

[0295] Ingredients Volume DNA mix X μl 153+181 Splint oligo (20 μM) 3 μl <![CDATA[NF-H2O]]> Make up to 50 μl Total volume 50 μl

[0296] Wherein, DNA mix is ​​the mixed DNA sample purified in the previous step. 153+181 Splint oligo: 5'-CGAGAACGTCTCCGTAGCCATGTC-3' (SEQ ID No. 21).

[0297] Table 9 Single-strand thermal denaturation reaction parameter settings

[0298] Cycle number Temperature Time 1 95℃ 3 min 1 4℃ Forever

[0299] 10. Cyclization reaction

[0300] The reaction system of the cyclization reaction is shown in Table 10.

[0301] Table 10 Reaction system of cyclization reaction

[0302] Ingredients Volume DNA mix from previous step 50 μl 10X TA buffer 6 μl 100 mM ATP 0.6 μl T4 DNA ligase (400 U / μl) 0.6 μl TE Buffer 2.8 μl Total volume 120 μl

[0303] Among them, the manufacturer's product number of 10×TA buffer is EPICENTRE TA6160.

[0304] After preparing the above reaction solution, vortex and centrifuge for 5 seconds; incubate at 37°C for 45 minutes and keep warm at 4°C.

[0305] 11. Enzyme digestion

[0306] The enzyme digestion reaction system is shown in Table 11.

[0307] Table 11 Enzyme digestion reaction system

[0308] Ingredients Volume Ligation product mix from previous step 60 μl 10X TA buffer 0.4 μl EXO I (20 U / μl) 1.95 μl EXO III (100 U / μl) 0.65 μl TE Buffer 1 μl Total volume 64 μl

[0309] Among them, the manufacturer's product number of 10×TA buffer is EPICENTRE TA6160.

[0310] After preparing the reaction solution, vortex and shake gently for 5 seconds; incubate at 37°C for 30 minutes and keep warm at 4°C. After the reaction is complete, add 4 μl of 0.1 mM EDTA (AMBION AM9260G) to terminate the reaction.

[0311] 12. Magnetic bead purification

[0312] 12.1 Take 90 μl of PEG32 beads and add them to the above reaction solution. Vortex to mix thoroughly and incubate at room temperature for 10 minutes.

[0313] 12.2 Briefly centrifuge the reaction tube and place it on a magnetic rack to separate the beads and liquid. Wait for the solution to clear (approximately 5 minutes) and carefully remove the supernatant.

[0314] 12.3 Keeping the EP tube in the magnetic rack, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads. Incubate at room temperature for 30 seconds and carefully remove the supernatant.

[0315] 12.4 Repeat the previous step, rinsing twice in total;

[0316] 12.5 Keep the EP tube in the magnetic rack and air-dry the magnetic beads for 3 minutes with the lid open;

[0317] 12.6 Remove the EP tube from the magnetic rack and add 32 μl of sterile ultrapure water for elution. Vortex or gently pipette to mix thoroughly. Briefly centrifuge the reaction tube and place it on a magnetic rack to separate the beads and liquid. Allow the solution to clear (approximately 2 minutes) and carefully pipette the supernatant into a sterile EP tube. Store at -20°C (this is the final library).

[0318] 12.7 After purification, take 1 μl of the product and measure the ssDNA concentration;

[0319] The result showed: 2.89ng / μl (*Experienced value: ssDNA concentration 0.6-3.0ng / ul, total yield 80-100ng)

[0320] 13. High-throughput sequencing

[0321] The samples were sequenced using BGI-SEQ500 (paired-end sequencing) using the PE50+26+10 sequencing method.

[0322] 14. Data Analysis

[0323] 2. Multidimensional Analysis of Multicellular Epigenomics

[0324] 1. Steps 1-6 are the same as steps 1-6 of a multidimensional analysis of single-cell epigenomics.

[0325] 7. Column purification (using the QIAamp DNA Mini Kit (Cat. No. 51304), which includes the PB Buffer, PE Buffer, and spin columns described below)

[0326] 7.1 Add 125 μl (5 times the volume) of PB Buffer to the sample after the reaction in step 6.3 and mix thoroughly by pipetting.

[0327] 7.2 Place a filter column in a 2 ml centrifuge tube and add the mixture from step 7.1 to the column.

[0328] 7.3 Place the column in the centrifuge tube and centrifuge at 13000 rpm for 1 minute until all sample has passed through the column, discard the waste.

[0329] 7.4 Add 750 μΐ of PE Buffer to the column and centrifuge at 13000 rpm for 1 minute, discard the waste.

[0330] 7.5 Leave the column empty for 1 minute to remove residual alcohol.

[0331] 7.6 Place the column in a clean 1.5 ml centrifuge tube.

[0332] 7.7 Add 20 μΐ of TE Buffer (AM9858) to elute the DNA, leave for 1 minute and then centrifuge at 13000 rpm for 1 minute to collect the liquid.

[0333] 8. PCR amplification

[0334] 8.1 Prepare the PCR Ready Mix according to the following Table 12:

[0335] Table 12 PCR Ready Mix preparation

[0336] Composition Amount (μl) 2X KAPA Ready Mix 25 Bulk-N5 Primer (20 μM) 2 183+C-pho Primer (20 μM) 2 7.7 step DNA 19

[0337] <![CDATA[NF-H2O]]> 2 Total volume 50

[0338] Bulk-N5 Primer: 5'-CGTAGCCATGTCGTTCTGCGTCGTCGGCAGCGTC-3' (SEQ ID No. 22);

[0339] 183+C-pho Primer: 5'-pho GAGACGTTCTCGACTCAGCAGAGTCTCGTGGGCTCGG-3' (SEQ ID No. 23).

[0340] 8.2 Vortex the reaction mix and centrifuge briefly;

[0341] 8.3 Run the reaction on a PCR machine according to the following Table 13 conditions with a hot lid temperature setting of 105°C.

[0342] Table 13 PCR program

[0343]

[0344] Note: The number of PCR cycles can be changed accordingly for different samples;

[0345] Stopping point: PCR products can be stored at 4°C for 24 hours. After PCR, use Qubit quantification to determine if there are any problems with the previous treatment. If the concentration is 5 ng / μl, it means that the sample treatment has failed and no further steps are required.

[0346] 9. Steps 9-14 are the same as steps 9-14 in step 1 for multi-dimensional analysis of single-cell epigenomics.

[0347] 3. Results and Analysis

[0348] The quality inspection results of DNA products obtained from single cells are as follows Figure 3 The quality inspection results of DNA products obtained from multiple cells are shown in Figure 4 As shown. Figure 3 and Figure 4 It can be seen that both the single-cell and multi-cell examples are enriched with fragments of 200-500 bp, proving that the experiment is feasible.

[0349] The results of single cell multi-omics sequencing analysis are shown in Table 13 and Figure 5 、 Figure 6 shown. Figure 5 The unique reads number obtained for single-cell CUT&Tag data. Figure 6 is the unique reads number obtained from single cell ATAC-seq data. Figure 5 、 Figure 6 It can be seen that: Table 13 shows the single cell data obtained in Example 1, the number of cells obtained and the total number of reads, to support the subsequent analysis. Figure 5 、 Figure 6 The numbers of reads captured by the CUT&Tag data and ATAC-seq data for each cell in Example 1 are shown separately. For the CUT&Tag data, more than 100 cells captured over 1000 reads, while for the ATAC-seq data, most cells captured over 5000 reads. The number of valid reads captured supports the subsequent analysis.

[0350] Table 13 Single-cell multi-omics sequencing results

[0351] CUT&Tag ATAC-seq Cell number 376 2308 Total read number 12308078 586454498

[0352] Read number on alignment 12008982 571379934 Read number alignment probability 97.57％ 97.43％ Q30 91.5％ 93.4％

[0353] Figure 7 and Figure 8 The fragment distribution is obtained from the single-cell omics CUT&Tag data and ATAC-seq data. Figure 7The results show that peaks of different fragment sizes obtained by single-cell CUT&Tag represent periodic changes in nucleosomes. This demonstrates that different nucleosome numbers can be obtained, verifying the accuracy of the experiment. Figure 8 Single-cell omics shows ATAC-seq data, with the main peak at the first one, indicating that ATAC mainly captures DNA in the open regions of chromosomes.

[0354] The results of single-cell multi-omics sequencing analysis are shown in Table 14. As shown in Table 14, Table 14 shows the multi-cell data obtained in Example 2, the number of cells obtained and the total number of reads to support the subsequent analysis.

[0355] Table 14 Multi-cell multi-omics sequencing results

[0356] CUT&Tag ATAC-seq Total read number 3982091 472527602 Read number on alignment 8821920 571379934 Read number on alignment probability 99.19％ 98.63％

[0357] Figure 9 Shows the distribution of fragments obtained by multi-cell CUT&Tag, Figure 10 Shows the fragment distribution obtained by multi-cell ATAC-seq. Figure 9 and Figure 10 It can be seen that Figure 9 and Figure 10 The fragment distributions obtained are multi-cell CUT&Tag data and ATAC-seq data. Figure 9 The results show that peaks of different fragment sizes obtained by multicellular CUT&Tag represent periodic changes in nucleosomes. This demonstrates that different nucleosome numbers can be obtained, verifying the accuracy of the experiment. Figure 10 The image shows multi-cell ATAC-seq data. The main peak is at the first one, indicating that ATAC mainly captures DNA in the open regions of chromosomes.

[0358] Figure 11 The peak distribution diagram of some CUT&Tag data is shown. The first red line (single cell CUT&Tag) represents the data obtained in step 1 of Example 1 of the present invention. The second black line in the middle (cells CUT&Tag) represents the data obtained in step 2. The third green line (Henikoff CUT&Tag) is the data obtained by single-omics CUT&Tag in the reference (https: / / doi.org / 10.1038 / s41467-019-09982-5). The fourth blue line (ChIP-seq) is the data from the ChIP-seq database. The peak distribution of the four data sets is basically consistent, proving that the CUT&Tag data obtained in steps 1 and 2 of Example 1 of the present invention are authentic and valid.

[0359] Figure 12The peak distribution diagram of ATAC-seq partial data is shown, the first red (single cell ATAC-seq) is the ATAC data obtained in step one of the embodiment 1 of the application, the second black (cells ATAC-seq) is the ATAC data obtained in step two, and the third blue (K562 ATAC-seq) is the ATAC-seq peak distribution of K562 cells in the database, the peak distributions of the three data are basically consistent, proving that the ATAC data obtained in the embodiment is true and effective.

[0360] Industrial applications

[0361] The application simultaneously realizes ChIP-seq and ATAC-seq sequencing in cells, ATAC-seq can provide information of open regions of cell chromatin, ChIP-seq can provide information of specific protein binding sequences, the combination of the two can obtain more abundant epigenetic information, which is beneficial for researchers to study developmental and disease-related cell population heterogeneity and draw cell atlas.

[0362] The method combines two dimensions of epigenomics, ATAC-seq and ChIP-seq, uses pA-Tn5 fusion protein (i.e. ChiTag transposase) and conventional Tn5 transposase to respectively embed different linker sequences in cells, and respectively analyzes chromatin open region information and specific protein binding sequence information at single cell level. The two dimensions of epigenomics at single cell level are jointly analyzed, and more abundant epigenomic information is obtained.

[0363] The application can be used for developing high-throughput ATAC-seq and ChIP-seq combined library kit for tumor samples and developmental and disease samples, and can be applied to research developmental and disease-related cell population heterogeneity and draw cell atlas by researchers in scientific research application, and provides more information in epigenetic regulation. The application also has broad application prospects in clinical aspects. Compared with the current mRNA-based transcriptome sequencing detection method, the method is based on chromatin genome in cell nucleus, and the detection object is relatively not easy to degrade, and has higher tolerance to sample quality. The method can analyze different clinically characterized tumor cells, which has important significance for studying tumor cell evolution and metastasis in clinic. The application can capture specific protein interaction DNA sequence information and chromatin open region information at single cell level and multi-cell level, so as to analyze cell epigenome from multiple aspects, and is used for studying epigenetic regulation mechanism of developmental and disease-related cells.

Claims

1. A method for multidimensionally analyzing cellular epigenomics for non-disease diagnosis and treatment purposes, comprising the following steps: using ChiTag transposase and Tn5 transposase in cells to embed different adapter sequences, respectively, to achieve co-analysis of chromatin open region information and specific protein binding sequence information at the cellular level; The method is method A or method B; Method A is a method for multi-dimensional analysis of single-cell epigenomics, comprising the following steps: (A1) Change the permeability of the test cells; (A2) Adding antibodies corresponding to specific proteins to the cells treated in step (A1) and incubating; (A3) Adding secondary antibodies to the cells treated in step (A2) for incubation; (A4) adding ChiTag transposase to the cells treated in step (A3) and incubating; (A5) adding a reaction reagent to the cells treated in step (A4) and incubating them, and then adding Tn5 transposase; (A6) After the reaction in step (A5), single-cell droplets are generated using a water-in-oil structure for intra-droplet amplification; (A7) breaking the emulsion, amplifying and purifying; (A8) Perform enzyme digestion to obtain the final library; (A9) performing high-throughput sequencing on the final library obtained in step (A8) to analyze the chromatin open region information and specific protein binding sequence information at the single-cell level; Method B is a method for multi-dimensional analysis of multi-cellular epigenomics, comprising the following steps: (B1) Change the permeability of the test cells; (B2) adding antibodies corresponding to specific proteins to the cells treated in step (B1) and incubating; (B3) Adding secondary antibodies to the cells treated in step (B2) for incubation; (B4) adding ChiTag transposase to the cells treated in step (B3) and incubating; (B5) adding a reaction reagent to the cells treated in step (B4) and incubating them, and then adding Tn5 transposase; (B6) Purification and amplification; (B7) Perform enzyme digestion to obtain the final library; (B8) Perform high-throughput sequencing on the final library obtained in step (B7) to analyze the chromatin open region information and specific protein binding sequence information at the multi-cellular level.

2. The method according to claim 1, wherein: In steps (A1) and (B1), the changing of the permeability of the test cells is achieved by resuspending the test cells in NP40-digoxigenin washing buffer; The NP40-digoxin wash buffer is obtained by adding 0.01% NP40 and 0.01% digoxin to the basic wash buffer; The composition of the basic washing buffer is as follows: 20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM spermidine; 1× protease inhibitor.

3. The method according to claim 2, wherein: Said (A1) and (B1) comprise: first resuspending 500,000 to 1,000,000 cells in 1 ml of said NP40-digoxigenin washing buffer, then centrifuging and discarding the supernatant, and adding 49 μl of said NP40-digoxigenin washing buffer supplemented with 1.5-2.5 mM EDTA and resuspending the cells; In steps (A2) and (B2), the antibody corresponding to the specific protein is directly added to the second cell resuspension obtained in steps (A1) and (B1).

4. The method according to any one of claims 1 to 3, characterized in that: In steps (A2) and (B2), the antibody corresponding to the specific protein is an H3K27me3 antibody.

5. The method according to any one of claims 1 to 3, characterized in that: In steps (A2) and (B2), the incubation is performed at 23-28°C for 2 hours or at 4°C for 10-12 hours.

6. The method according to any one of claims 1 to 3, characterized in that: In steps (A3) and (B3), washing and centrifugation steps are further included before adding the secondary antibody; the washing is performed using the NP40-digoxigenin washing buffer described in claim 3; and the centrifugation is performed at 600 g for 3 minutes.

7. The method according to claim 6, characterized in that: In steps (A3) and (B3), after the last centrifugation, the step of adding the NP40-digoxigenin washing buffer described in claim 3 to the precipitate to resuspend the cells is further included; and the secondary antibody is added to the obtained cell resuspension.

8. The method according to any one of claims 1 to 3, characterized in that: In steps (A3) and (B3), the incubation is performed at 23-28° C. for 30 min.

9. The method according to any one of claims 1 to 3, characterized in that: In steps (A3) and (B3), the incubation further includes the steps of centrifugation and washing; the centrifugation is performed at 600 g for 3 minutes; and the washing is performed using the NP40-digoxigenin washing buffer solution described in claim 3.

10. The method according to any one of claims 1 to 3, characterized in that: In steps (A4) and (B4), before adding the ChiTag transposase, the steps of centrifuging and resuspending the cells are further included; the centrifugation is performed at 600g for 3 minutes; the resuspended cells are resuspended in ChiTag transposase incubation buffer; The chitag transposase incubation buffer comprises the following components: 0.01% (volume percentage) NP40, 0.01% digoxin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM spermidine, and 1× protease inhibitor.

11. The method according to claim 10, characterized in that: In steps (A4) and (B4), PrimerA, ChIP-Tn5-PrimerB and ChIP-Tn5-PrimerC are added together with the ChiTag transposase; The PrimerA is a single-stranded DNA shown in SEQ ID No.1 with a phosphate group modified at the 5' end; the ChIP-Tn5-PrimerB is a mixture of the four single-stranded DNAs shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5; the ChIP-Tn5-PrimerC is a mixture of the four single-stranded DNAs shown in SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 and SEQ ID No.

9.

12. The method according to claim 11, wherein: In steps (A4) and (B4), the ChiTag transposase is added to the cell suspension resuspended with the chitag transposase incubation buffer; The ChiTag transposase was added at a ratio of 1 μL of pA-Tn5 adapter complex to 99 μL of the cell suspension; the pA-Tn5 adapter complex was obtained by uniformly mixing the ChiTag transposase and the ChIP double-stranded adapter mixture at a molar ratio of 1:1 and incubating at 25° C. for 1 hour; the final concentration of the ChiTag transposase in the pA-Tn5 adapter complex was 5.75 pmol / μL; and the ChIP double-stranded adapter mixture contained PrimerA, ChIP-Tn5-PrimerB, and ChIP-Tn5-PrimerC.

13. The method according to claim 12, wherein: In steps (A4) and (B4), the ChIP double-stranded adapter mixture is prepared according to a method comprising the following steps: The first step is to prepare the following reaction system: Reaction system I: Primer A and ChIP-Tn5-Primer B were mixed in equal moles; Reaction system II: the Primer A and the ChIP-Tn5-Primer C were mixed in equal moles; In the second step, the reaction system I and the reaction system II were subjected to the following reaction procedures: 75°C for 15 min; 60°C for 10 min; 50°C for 10 min; 40°C for 10 min; 25°C for 30 min; In the third step, after the reaction is completed, the reaction system I and the reaction system II are mixed in equal volumes to obtain the ChIP double-stranded adapter mixture.

14. The method according to any one of claims 1 to 3, characterized in that: In steps (A4) and (B4), the incubation is performed at 23-28° C. for 1 hour.

15. The method according to any one of claims 1 to 3, characterized in that: In steps (A4) and (B4), the incubation further includes the steps of centrifugation and washing; the centrifugation is performed at 300 g for 3 minutes; and the washing is performed using the chitag transposase incubation buffer as described in claim 10.

16. The method according to any one of claims 1 to 3, characterized in that: In steps (A5) and (B5), the reaction reagents are directly added to the cell pellet obtained after centrifugation in step (A4).

17. The method according to any one of claims 1 to 3, characterized in that: In steps (A5) and (B5), the reaction reagent is chitag transposase shearing buffer; The chitag transposase disruption buffer is a solution obtained by adding 10 mM MgCl2 to the chitag transposase incubation buffer described in claim 11.

18. The method according to any one of claims 1 to 3, characterized in that: In steps (A5) and (B5), the incubation is performed at 37° C. for 60 min.

19. The method according to claim 18, wherein: In steps (A5) and (B5), after the incubation, the mixture was centrifuged at 300 g for 3 min, and then Tn5 transposase was added and reacted at 37° C. and 500 rpm for 30 min.

20. The method according to any one of claims 1 to 3, characterized in that: In steps (A5) and (B5), primerA, ATAC-Tn5-primerB, and ATAC-Tn5-PrimerC are added together with the Tn5 transposase; The PrimerA is a single-stranded DNA shown in SEQ ID No. 1 with a phosphate group modified at the 5' end; The ATAC-Tn5-primerB is a mixture of the four single-stranded DNAs shown in SEQ ID No.10, SEQ ID No.11, SEQ ID No.12 and SEQ ID No.13; the ATAC-Tn5-PrimerC is a mixture of the four single-stranded DNAs shown in SEQ ID No.14, SEQ ID No.15, SEQ ID No.16 and SEQ ID No.

17.

21. The method according to claim 20, characterized in that: In steps (A5) and (B5), the Tn5 transposase is added in the form of an ATAC transposition mix; each 25 μl of the ATAC transposition mix contains 5 μl of 5X TAG buffer, 16 μl of PBS containing 1% BSA, and 4 μl of the Tn5 adaptor complex; the Tn5 adaptor complex is obtained by mixing Tn5 transposase and a Tn5 double-stranded adapter mixture at a molar ratio of 1:1, and incubating at 25°C for 1 hour, wherein the final concentration of Tn5 transposase in the Tn5 adaptor complex is 0.875 U / μl; the Tn5 double-stranded adapter mixture contains PrimerA, ATAC-Tn5-primerB, and ATAC-Tn5-PrimerC.

22. The method according to claim 21, characterized in that: The Tn5 double-stranded adapter mixture is prepared according to a method comprising the following steps: The first step is to prepare the following reaction system: Reaction system I: Primer A and ATAC-Tn5-primer B were mixed in equal moles; Reaction system II: the Primer A and the ATAC-Tn5-Primer C were mixed in equal moles; In the second step, the reaction system I and the reaction system II were subjected to the following reaction procedures: 75°C for 15 min; 60°C for 10 min; 50°C for 10 min; 40°C for 10 min; 25°C for 30 min; In the third step, after the reaction is completed, the reaction system I and the reaction system II are mixed in equal volumes to obtain the Tn5 double-stranded adapter mixture.

23. The method according to any one of claims 1 to 3, characterized in that: In step (A6), the primers used for the in-droplet amplification are: Tn-Primer and 183+C Primer; the Tn-Primer is the single-stranded DNA shown in SEQ ID No. 18, and the 183+C Primer is the single-stranded DNA shown in SEQ ID No.

19.

24. The method according to any one of claims 1 to 3, characterized in that: In step (A7), the primers used for the amplification are: Tn Primer and 183-pho Primer; the Tn-Primer is the single-stranded DNA shown in SEQ ID No. 18, and the 183-pho Primer is the single-stranded DNA shown in SEQ ID No. 20 with a phosphate group modified at the 5' end.

25. The method according to any one of claims 1 to 3, characterized in that: In step (A7), the purification is performed by adding 1.2 times the volume of magnetic beads to the amplified product for purification.

26. The method according to claim 25, characterized in that: In step (A7), the magnetic beads are AgencourtAMPure XP magnetic beads.

27. The method according to any one of claims 1 to 3, characterized in that: In step (B6), the primers used for the amplification are Bulk-N5 Primer and 183+C-pho Primer; the Bulk-N5 Primer is the single-stranded DNA shown in SEQ ID No. 22; the 183+C-pho Primer is the single-stranded DNA shown in SEQ ID No. 23 with a phosphate group modified at the 5' end.

28. The method according to any one of claims 1 to 3, characterized in that: In steps (A8) and (B7), a cyclization step is further included before the enzyme cleavage; The cyclization is achieved with the aid of 153+181 Splint oligo; The 153+181splint oligo is a single-stranded DNA shown in SEQ ID No.

21.

29. The method according to any one of claims 1 to 3, characterized in that: In steps (A8) and (B7), the enzyme digestion is performed using EXO I enzyme and EXO III enzyme.

30. The method according to any one of claims 1 to 3, characterized in that: In steps (A8) and (B7), after the enzymatic digestion, the method further includes a step of purifying the enzymatic digestion products with magnetic beads.

31. The method according to claim 30, wherein: In steps (A8) and (B7), the magnetic beads used to purify the enzyme-digested products are PEG32 beads.

32. The method according to any one of claims 1 to 3, characterized in that: In steps (A9) and (B8), the sequencing is high-throughput sequencing.

33. The method according to claim 32, wherein: The high-throughput sequencing is double-end sequencing, and the sequencing method is PE50+26+10.

34. A method for constructing a DNA library for multidimensional analysis of cellular epigenomics, comprising steps (A1) to (A8) of method A according to any one of claims 1 to 31 or steps (B1) to (B7) of method B according to any one of claims 1 to 31.

35. Use of the method according to any one of claims 1 to 34 for non-disease diagnosis and treatment purposes in any of the following: (C1) Studying cell population heterogeneity related to development and / or disease; (C2) Draw a cell map; (C3) Analyze tumor cells with different clinical manifestations; (C4) Clinically studying tumor cell evolution and / or metastasis.

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