Preparation method of single-cell CUTtag library

Abstract

The present application provides a single cell CUTamp; the Tag library preparation method comprises the following steps: (a) obtaining single cells by using a flow cytometer or microdissection, carrying out cross-linking immobilization by using formaldehyde, and terminating the reaction by using glycine; (b) incubating the cells by using a low-salt buffer solution containing Digionin (digitonin) to increase the permeability of a membrane, so that macromolecular substances such as an antibody and the like can enter cell nucleuses; (c) blocking the non-specific sites in the nucleus by using BSA (Bovine Serum Albumin); (d) calculating the optimal antibody dosage by using an antibody calculation formula, incubating the primary antibody in combination with gradient temperature, and maximally ensuring the combination of the primary antibody and the target protein; (e) adding a second antibody corresponding to the first antibody for incubation; (f) adding pA-Tn5 transposase, and activating the transposase to react by using Mg + 2 ions; (g) adding EDTA (Ethylene Diamine Tetraacetic Acid), SDS (Sodium Dodecyl Sulfate), protease K and the like to terminate the reaction, and purifying by using a And (h) monitoring an amplification curve by using real-time quantitative PCR to determine an optimal amplification cycle number, amplifying by using the optimal amplification cycle number, and performing fragment screening and purification on a PCR product to obtain a final library.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically, to a method for preparing a single-cell CUT&Tag library. Background Art

[0002] The CUT&Tag (Cleavage Under Targets and Tagmentation) technique is a technique for studying the interaction between proteins and DNA. A fusion protein of Tn5 transposase and protein A (pA-Tn5) is used, which is combined with an antibody to activate pA-Tn5 to cause targeted factor labeling and generate DNA sequencing fragments. Due to its advantages such as low background, short experimental time, and low sample volume, it is increasingly widely used in the field of studying the interaction between proteins and DNA. Single-cell CUT&Tag is a technique for studying chromatin states and transcription factor binding at the single-cell level. It combines the advantages of the CUT&Tag technique and can analyze chromatin modifications and protein-DNA interactions at single-cell resolution. Single-cell CUT&Tag has a wide range of applications: it can reveal chromatin modification differences in different cell types or states; it can be used to study the dynamic changes of chromatin states during development; in diseases such as cancer, it can help identify epigenetic changes related to the disease; it can analyze the binding patterns of specific transcription factors in different cell types, etc.

[0003] Currently, kits for preparing CUT&Tag high-throughput libraries on the market include those from Novoprotein Scientific Inc. These kits are only for constructing libraries of samples with a large number of cells. When used for single-cell CUT&Tag library construction, it is extremely easy to fail in experimental steps such as antibody binding, antibody dosage, transposase treatment, and PCR amplification, resulting in a very low success rate of single-cell CUT&Tag library construction. Therefore, a method for preparing a single-cell CUT&Tag library is needed. Summary of the Invention

[0004] The purpose of the present invention is to perform CUT&Tag experiments at the single-cell level for accurately detecting the binding sites of proteins on chromatin.

[0005] The present application provides a method for preparing a single-cell CUT&Tag library, and the method includes:

[0006] (a) Capture and fixation of single cells;

[0007] (b) Permeation of cell membranes and nuclear membranes;

[0008] (c) Blocking and washing;

[0009] (d) Incubation with primary antibody;

[0010] (e) Incubation with secondary antibody;

[0011] (f) Incubation and activation with pA-Tn5 transposase;

[0012] (g) Termination of the reaction and DNA purification;

[0013] (h) Perform PCR amplification to obtain a sequencing library.

[0014] In some embodiments, in step (a), single cells are captured by flow cytometry or microdissection, formaldehyde is used for fixation and crosslinking, and glycine is used to terminate the crosslinking reaction to prevent over-crosslinking;

[0015] In some embodiments, in step (b), cells are incubated with a low-salt buffer containing Digitonin to increase membrane permeability, allowing macromolecules such as antibodies to enter the cell nucleus;

[0016] In some embodiments, in step (c), BSA is used to block non-specific sites in the cell nucleus;

[0017] In some embodiments, in step (d), the usage amount (Y Ab， (unit: μg) of the primary antibody is calculated by the following formula:

[0018]

[0019] where P a is the relative abundance of the target protein, A f is the antibody affinity, S p is the antibody specificity, V e is the reaction volume, I f is the incubation factor, O f is the optimization coefficient;

[0020] Furthermore, P a is the relative abundance of the target protein, which is the relative abundance of the target protein to histone H3; V e is the reaction volume, unit: L; I f is the incubation factor, which is 1 when the incubation time of the primary antibody is within 0 - 2 h, and 0.9 when the incubation time of the primary antibody is greater than 2 h; O f is the optimization coefficient, calculated by regression analysis to be 3.54;

[0021] Furthermore, the primary antibody incubation is carried out with gradient temperature incubation, incubating at 4°C for 1 h and then transferring to 37°C for 1 h;

[0022] In some embodiments, in step (e), add the secondary antibody corresponding to the primary antibody for incubation;

[0023] In some embodiments, in step (f), pA-Tn5 transposase is added and the transposase reaction is activated with Mg +2 ions;

[0024] In some embodiments, in step (g), EDTA, SDS, proteinase K, etc. are added to terminate the reaction, and a micro DNA purification kit is used for purification;

[0025] Furthermore, during the final elution of DNA in the purification using the micro DNA purification kit, a small volume (5 μl) is used;

[0026] In some embodiments, in step (h), real-time quantitative PCR is used to monitor the amplification curve to determine the optimal number of amplification cycles;

[0027] Furthermore, amplification is stopped within 2 cycles after the amplification curve enters the plateau phase to prevent over-amplification;

[0028] Furthermore, the amplified product is screened by fragment size to obtain the final library, and the screened fragment size is 200 - 500 bp.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] (1) High success rate. By using the antibody calculation formula to calculate the optimal antibody dosage and incubating the primary antibody at gradient temperatures, the binding of the primary antibody to the target protein is maximally ensured, greatly increasing the success rate of single-cell CUT&Tag library construction.

[0031] (2) High accuracy. By adding a blocking step before primary antibody incubation, the non-specific binding of the primary antibody is reduced. Using real-time quantitative PCR to monitor the amplification curve to determine the optimal number of amplification cycles reduces the introduction of PCR bias. The fragment screening of PCR products removes the influence of large fragments on the sequencing results. The combination of multiple methods greatly improves the accuracy of sequencing data.

[0032] (3) Simple operation. Compared with conventional CUT&Tag, there is no need for new instruments, and the experimental steps are relatively simple and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] When reading in conjunction with the following attached Figure 1 drawings, the above and other features of the present application will be more fully described. It can be understood that these drawings only depict several embodiments of the present application, and thus should not be considered as limiting the scope of the present application. By using the drawings, the present application will be more clearly and detailedly described.

[0034] Figure 1 For the calculation data of the O f value in Example 1 of the present application.

[0035] Figure 2 O regression curve for Example 1 of the present application f value

[0036] Figure 3 A1 library quality inspection chart for Example 2 of the present application

[0037] Figure 4 Library quality inspection chart for Example 3 of the present application Detailed implementation manners

[0038] The following examples are described to assist in understanding the present application, and the examples are not and should not be construed in any way as limiting the scope of protection of the present application.

[0039] For the experimental methods without specific conditions indicated in the following examples, they are carried out according to conventional experimental conditions, such as the conditions described in Molecular Cloning: A Laboratory Manual by Sambrook et al. (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise specified, the materials used in the examples are all commercially available products.

[0040] Example 1:

[0041] (1) Sort single Hela cells into a 0.2 ml low-attachment PCR tube using a flow cytometer, and 2 μl of PBS is pre-added to the tube.

[0042] (2) Add 2 μl of 2% formaldehyde solution to the cells obtained in step (1), mix well and incubate at room temperature for 5 minutes for mild fixation, then add 1 μL of 1 M glycine to terminate the fixation reaction, and incubate at room temperature for 5 minutes.

[0043] (3) Add 10 μl of Wash buffer (20 mM HEPES, pH 7.5;

[0044] 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail) to the cells obtained in step (2), mix well, and then centrifuge at 600 g for 3 min at room temperature.

[0045] (4) Repeat step (3) once, and then add 10 μl of Wash buffer to resuspend the cells.

[0046] (5) Take 1 μl of commercially purchased ConA beads, then add 20 μl of Wash buffer, mix well, and then place on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard all the supernatant, and then resuspend the magnetic beads with 10 μl of Wash buffer.

[0047] (6) Add the magnetic beads obtained in step (5) to the 10 μl of cell suspension obtained in step (4), carefully mix well, and incubate with rotation at room temperature for 15 min.

[0048] (7) Place the incubation product of step (6) on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard the supernatant completely.

[0049] (8) Resuspend the mixture of magnetic beads and cells obtained in step (7) with 10 μl of Dig-wash buffer containing 2 mM EDTA (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail; 0.05% Digitonin).

[0050] (9) Add 10 μL of blocking buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM

[0051] Spermidine; 1× Protease inhibitor cocktail; 0.1% BSA; 0.01% Digitonin),

[0052] Incubate at room temperature for 10 minutes to prevent non-specific binding.

[0053] (10) Gently wash the cells with 10 μl of Wash buffer and repeat twice to remove unbound antibodies. (11) The formula for the amount of antibody used is: where P a is the relative abundance of the target protein, A f is the antibody affinity, S p is the antibody specificity, V e is the reaction volume, I f is the incubation factor, O f is the optimization coefficient, where P a , A f , S p can be confirmed by querying the instruction manual, etc. after determining the target protein and the antibody used. V e and I f are confirmed according to the experimental procedures. Only the value of O f needs to be calculated from experimental data. In order to determine O fFor the values, we used data of 11 antibodies (H3K27me3, H3K27ac, H3K4me3, H3K4ac, MYC, CTCF, EP300, SP1, NFKB, GATA3, SOX2). For each antibody, gradient experiments were conducted with different antibody dosages as one group. Different amounts of antibodies were added to 11 groups of samples.

[0054] (12) Incubate the samples at 4°C for 1 h and then transfer them to 37°C for 1 h.

[0055] (13) Place the complex obtained in step (12) on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard the supernatant completely.

[0056] (14) Dilute the appropriate secondary antibody with Dig-wash buffer at a ratio of 1:50. Then resuspend the complex obtained in step (13) with the diluted secondary antibody and incubate with rotation at room temperature for 1 h.

[0057] (15) Place the mixture obtained by incubation in step (14) on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard the supernatant completely.

[0058] (16) Then add 10 μl of Dig-wash buffer to resuspend the cells in the mixture of step (15), mix well, and then place on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard the supernatant completely.

[0059] (17) Repeat step (16) twice.

[0060] (18) Dilute the pA-Tn5 complex with Dig-med Buffer (0.05% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl,

[0061] 0.5 mM Spermidine, 1× Protease inhibitor cocktail) to a final concentration of 0.4 μM. Then resuspend the mixture obtained in step (17), mix well, and incubate at room temperature for 1 h.

[0062] (19) Place the mixture obtained in step (18) on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard the supernatant completely.

[0063] (20) Add Dig-med Buffer to the mixture obtained in step (19), mix well, place on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard the supernatant completely.

[0064] (21) Repeat step (20) twice.

[0065] (22) Add 1 μl of Dig-med containing 100 mM MgCl2 to the mixture obtained in step (21), mix well, and incubate at 37 °C for 1 h.

[0066] Buffer, mix well, and incubate at 37 °C for 1 h.

[0067] (23) Add 2 μl of 0.5 M EDTA, 1 μl of 10% SDS, and 1 μl of 20

[0068] mg / ml Proteinase K to the mixture in step (22) to terminate the fragmentation reaction. Then incubate at 55 °C for 30 min or overnight at 37 °C.

[0069] (24) Use the MinElute PCR Purification Kit for DNA extraction and elute with a 5 μl volume.

[0070] (25) Directly perform PCR amplification on the DNA obtained in step (24), 12.5 μl of SYBR Green Master Mix, 1 μl of primer1 (TD202-AC, Vazyme), 1 μl of primer2 (TD202-AK, Vazyme),

[0071] Make up the volume with 5.5 μl of water. After mixing, perform qPCR reaction, react at 72 °C for 5 min, pre-denature at 98 °C for 30 s,

[0072] Denature at 98 °C for 10 s, anneal at 60 °C for 30 s, extend at 72 °C for 30 s, monitor the amplification curve in real time, stop the cycle within 2 cycles after the amplification curve enters the plateau phase to prevent over-amplification, extend at 72 °C for 5 min,

[0073] Store at 4 °C.

[0074] (26) Screen and purify the PCR products obtained in step (25). First, use a 0.6x AMPure XP magnetic bead ratio to remove fragments larger than 500 bp, and then use a 0.3x AMPure XP magnetic bead ratio to recover

[0075] the target fragments of 200 - 500 bp.

[0076] (27) After performing fragment length detection by Agilent 2100 Bioanalyzer and concentration quantification by Invitrogen Qubit, it can be used for sequencing on the Illumina platform to obtain sequencing data. According to the library quality inspection results (Qubit concentration greater than 1 ng / μl and fragment length range of 200 - 500 bp) and the sequencing data quality (Q30 greater than 85%), the optimal dosages of 11 antibodies were determined. Substitute each value into the formula and use the regression algorithm to calculate Of The value is 3.54, Figure 1 is O f Calculation data of the value, Figure 2 is O f Regression curve of.

[0077] Example 2:

[0078] (1) Use a flow cytometer to sort single A549 cells into 0.2 ml low-attachment PCR tubes. 2 μl of PBS has been pre-added to the tubes. Sort 4 tubes and label them as A1, A2, A3, and A4 respectively.

[0079] (2) Add 2 μl of 2% formaldehyde solution to 3 of the tubes of cells (A1 - A3) obtained in step (1). After mixing, incubate at room temperature for 5 minutes for mild fixation. Add 1 μL of 1 M glycine to terminate the fixation reaction and incubate at room temperature for 5 minutes.

[0080] (3) Add 10 μl of Wash buffer

[0081] (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1×Protease

[0082] inhibitor cocktail) to the 3 tubes of cells obtained in step (2) and A4 obtained in step (1). Mix well, then centrifuge at 600 g for 3 min at room temperature.

[0083] (4) Repeat step (3) once, then add 10 μl of Wash buffer to resuspend the cells.

[0084] (5) Take 1 μl of commercially purchased ConA beads, then add 20 μl of Wash buffer, mix well, then place on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard all the supernatant, and then resuspend the magnetic beads with 10 μl of Wash buffer.

[0085] (6) Add the magnetic beads obtained in step (5) to the 10 μl of cell suspension obtained in step (4), carefully mix well, and rotate and incubate at room temperature for 15 min.

[0086] (7) Place the incubation product of step (6) on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard all the supernatant.

[0087] Resuspend the mixture of magnetic beads and cells obtained in step (7) with 10 μl of Dig-wash buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail; 0.05% Digitonin) containing 2 mM EDTA.

[0088] (9) Add 10 μL of blocking buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail; 0.1% BSA; 0.01% Digitonin), and incubate at room temperature for 10 minutes to prevent non-specific binding.

[0089] (10) Gently wash the cells with 10 μL of Wash buffer twice to remove unbound antibodies.

[0090] (11) Calculate the amount of antibody used using the primary antibody calculation formula. P of H3K27me3 antibody a is 10%, A f is 1X10 9 , S p is 0.94, V e is 1X10 -5 , I f is 1, O f is 3.54, and it is calculated that Y Ab is 3.8X10 -5 ug, while the amount of antibody corresponding to a single cell calculated according to the H3K27me3 antibody instruction manual is 5X10 -6 ug. Add 3.8 μl of the antibody diluted to 0.01 ng / μl to the three samples A1, A2, and A4, and add 0.5 μl of the antibody diluted to 0.01 ng / μl to sample A3.

[0091] (12) Incubate samples A1, A3, and A4 at 4 °C for 1 h and then at 37 °C for 1 h, and incubate sample A2 at 37 °C for 2 h.

[0092] (13) Place the complex obtained in step (12) on a magnetic stand (or centrifuge at low speed), and discard the supernatant after the solution becomes clear.

[0093] (14) Dilute the appropriate secondary antibody with Dig-wash buffer at a ratio of 1:50. Then resuspend the complex obtained in step (13) with the diluted secondary antibody and incubate with rotation at room temperature for 1 h.

[0094] (15) Place the mixture obtained from step (14) on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard the supernatant completely.

[0095] (16) Then add 10 μl of Dig-wash buffer to resuspend the cells in the mixture from step (15). Mix well, then place on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard the supernatant completely.

[0096] (17) Repeat step (16) twice.

[0097] (18) Dilute the pA-Tn5 complex with Dig-med Buffer (0.05% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1× Protease inhibitor cocktail) to a final concentration of 0.4 μM. Then resuspend the mixture obtained from step (17), mix well, and incubate at room temperature for 1 h.

[0098] (19) Place the mixture obtained from step (18) on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard the supernatant completely.

[0099] (20) Add Dig-med Buffer to the mixture obtained from step (19). Mix well, place on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard the supernatant completely.

[0100] (21) Repeat step (20) twice.

[0101] (22) Add 1 μl of Dig-med Buffer containing 100 mM MgCl2 to the mixture obtained from step (21). Mix well and incubate at 37 °C for 1 h.

[0102] (23) Add 2 μl of 0.5 M EDTA, 1 μl of 10% SDS and 1 μl of 20

[0103] mg / ml Proteinase K to the mixture in step (22) to terminate the fragmentation reaction. Then incubate at 55 °C for 30 min or overnight at 37 °C.

[0104] (24) Use the MinElute PCR Purification Kit to extract DNA and elute with a 5 μl volume.

[0105] (25) Perform PCR amplification directly using the DNA obtained in step (24). Add 12.5 μl of SYBR Green Master Mix, 1 μl of primer 1, 1 μl of primer 2, and make up the volume to 5.5 μl with water. After mixing, perform qPCR reaction. React at 72 °C for 5 min, pre-denature at 98 °C for 30 s, denature at 98 °C for 10 s, anneal at 60 °C for 30 s, extend at 72 °C for 30 s, monitor the amplification curve in real time, stop the cycle within 2 cycles after the amplification curve enters the plateau phase to prevent over-amplification, extend at 72 °C for 5 min, and store at 4 °C.

[0106] (26) Screen and purify the PCR products obtained in step (25). First, use 0.6x AMPure XP magnetic beads to remove fragments larger than 500 bp, and then use 0.3x AMPure XP magnetic beads to recover the target fragments of 200 - 500 bp.

[0107] (27) After performing fragment length detection by Agilent 2100 Bioanalyzer and concentration quantification by Invitrogen Qubit, it can be used for sequencing on the Illumina platform to obtain sequencing data. Library A1 was successfully constructed, and the fragment length distribution is shown in the appendix Figure 3 , while libraries A2, A3, and A4 all failed to be constructed.

[0108] (28) Sequence library A1 on the Illumina Novaseq instrument using PE150 sequencing. The data obtained was analyzed using standard analysis (with the reference genome being GRCh38), and its duplication rate was 62%, showing a significant improvement compared to the 80% - 90% in current papers.

[0109] Example 3:

[0110] (1) Use a flow cytometer to sort single K562 cells into a 0.2 ml low-attachment PCR tube, and pre-add 2 μl of PBS to the tube.

[0111] (2) Add 2 μl of 2% formaldehyde solution to the cells obtained in step (1), mix well, incubate at room temperature for 5 minutes for mild fixation, add 1 μL of 1 M glycine to terminate the fixation reaction, and incubate at room temperature for 5 minutes.

[0112] (3) Add 10 μl of Wash buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail) to the cells obtained in step (2), mix well, then centrifuge at 600 g for 3 min at room temperature.

[0113] (4) Repeat step (3) once, then add 10 μl of Wash buffer to resuspend the cells.

[0114] (5) Take 1 μl of commercially purchased ConA beads, then add 20 μl of Wash buffer, mix well, then place on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard all the supernatant, and then resuspend the magnetic beads with 10 μl of Wash buffer.

[0115] (6) Add the magnetic beads obtained in step (5) to the 10 μl of cell suspension obtained in step (4), mix gently, and incubate with rotation at room temperature for 15 min.

[0116] (7) Place the incubation mixture from step (6) on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard all the supernatant.

[0117] (8) Resuspend the mixture of magnetic beads and cells obtained in step (7) with 10 μl of Dig-wash buffer containing 2 mM EDTA (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail; 0.05% Digitonin).

[0118] (9) Add 10 μL of blocking buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail; 0.1% BSA; 0.01% Digitonin), and incubate at room temperature for 10 minutes to prevent non-specific binding.

[0119] (10) Gently wash the cells with 10 μl of Wash buffer twice to remove unbound antibodies.

[0120] (11) Calculate the amount of antibody used using the primary antibody calculation formula. The P of the H3K4ac antibody a is 8%, the A f is 1X10 9 , the S p is 0.83, the V e is 1X10 -5 , the I f is 1, the O f is 3.54. Calculate that Y Ab is 3.4X10 -5 μg, and add 3.4 μl of the antibody diluted to 0.01 ng / μl to the sample.

[0121] Incubate the sample at 4 °C for 1 h and then transfer it to 37 °C for 1 h.

[0122] (13) Place the complex obtained in step (12) on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard the supernatant completely.

[0123] (14) Dilute the appropriate secondary antibody with Dig-wash buffer at a ratio of 1:50. Then resuspend the complex obtained in step (13) with the diluted secondary antibody and incubate with rotation at room temperature for 1 h.

[0124] (15) Place the mixture obtained by incubation in step (14) on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard the supernatant completely.

[0125] (16) Then add 10 μl of Dig-wash buffer to the mixture in step (15) to resuspend the cells, mix well, and then place it on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard the supernatant completely.

[0126] (17) Repeat step (16) twice.

[0127] (18) Dilute the pA-Tn5 complex with Dig-med Buffer (0.05% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl,

[0128] 0.5 mM Spermidine, 1× Protease inhibitor cocktail) to a final concentration of 0.4 μM. Then resuspend the mixture obtained in step (17), mix well, and incubate at room temperature for 1 h.

[0129] (19) Place the mixture obtained in step (18) on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard the supernatant completely.

[0130] (20) Add Dig-med Buffer to the mixture obtained in step (19), mix well, place it on a magnetic stand (or centrifuge at low speed). After the solution becomes clear, discard the supernatant completely.

[0131] (21) Repeat step (20) twice.

[0132] (22) Add 1 μl of Dig-med

[0133] Buffer containing 100 mM MgCl2 to the mixture obtained in step (21), mix well, and incubate at 37 °C for 1 h.

[0134] (23) Add 2 μl of 0.5 M EDTA, 1 μl of 10% SDS and 1 μl of 20

[0135] mg / ml Proteinase K to terminate the fragmentation reaction. Then incubate at 55 °C for 30 min or overnight at 37 °C.

[0136] (24) Use the MinElute PCR Purification Kit for DNA extraction and elute with a volume of 5 μl.

[0137] (25) Directly perform PCR amplification using the DNA obtained in step (24), 12.5 μl SYBR Green Master Mix, 1 μl primer1, 1 μl primer2, and make up the volume with 5.5 μl of water. After mixing, perform qPCR reaction: react at 72 °C for 5 min, pre-denature at 98 °C for 30 s, denature at 98 °C for 10 s, anneal at 60 °C for 30 s, extend at 72 °C for 30 s, monitor the amplification curve in real time, stop the cycle within 2 cycles after the amplification curve enters the plateau phase to prevent over-amplification, extend at 72 °C for 5 min, and store at 4 °C.

[0138] (26) Screen and purify the PCR products obtained in step (25). First, use a 0.6x AMPure XP magnetic bead ratio to remove fragments larger than 500 bp, and then use a 0.3x AMPure XP magnetic bead ratio to recover the target fragments of 200 - 500 bp.

[0139] (27) After performing fragment length detection by Agilent 2100 Bioanalyzer and concentration quantification by Invitrogen Qubit, it can be used for sequencing on the Illumina platform to obtain sequencing data. For library quality inspection, see the appendix Figure 4 。

[0140] Although the present application has disclosed multiple aspects and embodiments, other aspects and embodiments will be obvious to those skilled in the art. Without departing from the concept of the present application, several modifications and improvements can still be made, and these all fall within the protection scope of the present application. The multiple aspects and embodiments disclosed in the present application are only for illustrative purposes and are not intended to limit the present application. The actual protection scope of the present application is subject to the claims.

Claims

1. A method for preparing a single-cell CUT&Tag library, characterized in that, The method includes: (a) Capture and fixation of single cells: Capture single cells by flow cytometry or microdissection and perform mild cross-linking fixation; (b) Permeation of cell membrane and nuclear membrane: Incubate the cells with a low-salt buffer containing Digitonin to increase membrane permeability, allowing macromolecules such as antibodies to enter the nucleus; (c) Blocking and washing: Use BSA to block non-specific sites in the nucleus; (d) Primary antibody incubation: The usage amount (Y Ab， unit: μg) of the primary antibody is calculated by the following formula: Where P a is the relative abundance of the target protein, which is the relative abundance of the target protein to histone H3; A f is the antibody affinity; S p is the antibody specificity; V e is the reaction volume, with the unit of L; I f is the incubation factor, which is 1 when the incubation time of the primary antibody is within 0 - 2 h and 0.9 when the incubation time of the primary antibody is greater than 2 h; O f is the optimization coefficient, which is calculated to be 3.54 according to regression analysis; the primary antibody incubation is carried out at gradient temperatures, incubated at 4°C for 1 h and then transferred to 37°C for 1 h; (e) Incubation with secondary antibody: Add the secondary antibody corresponding to the primary antibody for incubation; (f) Incubation and activation of pA-Tn5 transposase: Add pA-Tn5 transposase and activate the transposase reaction with Mg +2 ions; (g) Termination of reaction and DNA purification: Add EDTA, SDS, proteinase K, etc. to terminate the reaction, and use a micro DNA purification kit for purification; (h) Perform PCR amplification to obtain a sequencing library: Use real-time quantitative PCR to monitor the amplification curve to determine the optimal number of amplification cycles, stop the cycle within 2 cycles after observing that the amplification curve enters the plateau phase to prevent over-amplification, and the amplified product is purified by fragment screening to obtain the final library, and the screened fragment size is 200-500bp.

2. The single-cell CUT&Tag library preparation method according to claim 1, characterized in that, The addition in step (c) reduces the binding of antibodies to non-specific sites and improves the accuracy of the library sequencing results.

3. The single-cell CUT&Tag library preparation method according to claim 1, characterized in that In step (d), the primary antibody incubation is performed using gradient temperature incubation, incubating at 4°C for 1h and then transferring to 37°C for 1h.

4. The single-cell CUT&Tag library preparation method according to claim 1, wherein, In step (g), the elution volume is 5ul when using a micro DNA extraction kit.

5. The method for preparing a single-cell CUT&Tag library according to claim 1, wherein, In step (h), use fluorescence quantitative PCR for amplification and monitor its amplification curve, and stop the cycle within 2 cycles after the amplification curve reaches the plateau phase. This can prevent sequencing result deviation caused by over-amplification.

6. The method for preparing a single-cell CUT&Tag library according to claim 1, wherein In step (h), the amplified product is purified by fragment screening, and the product with a length of 200-500bp is screened. The screening of fragment length can further ensure the quality of sequencing.

7. A calculation method for the dosage of the primary antibody in single-cell CUT&Tag, characterized in that, Composed of the relative abundance P of the target protein a , the antibody affinity A f , the antibody specificity S p , the reaction volume V e , the incubation factor I f and the optimization coefficient O f . Its calculation formula is: Where P a is the relative abundance of the target protein, which is the abundance of the target protein relative to histone H3, V e is the reaction volume, with the unit of L, I f is the incubation factor. When the incubation time of the primary antibody is within 0 - 2 h, it is 1; when the incubation time of the primary antibody is greater than 2 h, it is 0.9, O f is the optimization coefficient, which is calculated to be 3.54 according to regression analysis.

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