A composition and a method for constructing a library of genes interacting with proteins-DNA

Abstract

The application provides a composition and a construction method of a protein-DNA interaction gene library, and belongs to the technical field of biotechnology. The composition can be used as a transposase incubation buffer. By reducing the concentration of salt ions in the composition, the chromatin accessibility is improved. The protein-DNA interaction gene library constructed by the method has not only a significantly improved small fragment ratio, but also improved detection effect on transcription factor and excitatory histone modification target.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to a composition and a method for constructing a gene library for studying protein-DNA interactions. Background Technology

[0002] Chromatin immunoprecipitation (ChIP) is a widely used method for studying protein-DNA interactions, typically used to study transcription factor binding sites or histone-specific modification sites. Combining ChIP with massively parallel DNA sequencing allows researchers to precisely map the global DNA binding sites of proteins of interest. The basic ChIP process is as follows: (1) Crosslinking: Tissues or cells are fixed with formaldehyde to tightly bind DNA and proteins; (2) Fragmentation: This process destroys chromatin, ultimately obtaining DNA fragments and protein complexes for ChIP analysis; (3) Chromatin immunoprecipitation: Antibodies against the target protein are added to bind the target protein-DNA complex; (4) DNA recovery and purification: The purified and enriched DNA fragments are recovered and analyzed using downstream detection technologies (quantitative PCR, gene chips, sequencing, etc.) to obtain DNA sequence information specifically bound to the target protein (Park P.J., et al. ChIP-seq: advantages and challenges of a maturing technology. Nat RevGenet. 2009; 10:669-680). Figure 1 However, because ChIP technology requires formaldehyde cross-linking of tissues or cells before DNA fragmentation, this technology requires extremely high initial cell input (millions of cells), and is likely to produce false positives due to excessive formaldehyde cross-linking.

[0003] To overcome the technical limitations of ChIP, researchers have developed CUT&Run (Cleavage UnderTargets & Release Using Nuclease) and CUT&Tag (Cleavage UnderTargets & Tagmentation) technologies through continuous updates and optimizations. CUT&Tag and CUT&Run are techniques for studying protein-DNA interactions in living cells (Skene, PJ & Henikoff, S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. 2017; eLife 6, e21856.; Kaya-Okur HS., et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun. 2019; 10(1):1930.). They utilize antibody enrichment of TN5 transposase or Mnase nuclease to specifically cleave DNA near the target protein. Then, by constructing a library and sequencing the labeled DNA after cleavage, the global DNA binding sites of the target protein can be mapped. Figure 2 This technology, because it does not require cross-linking or physical DNA disruption, mitigates to some extent the drawbacks of ChIP technology, such as false positives and high cell input requirements.

[0004] Although CUT&Tag and CUT&Run technologies offer significant improvements over ChIP, they rely on the binding of the PA / PG-TN5 / MNase fusion protein to the Fc region of the secondary antibody, followed by the delivery of TN5 / MNase to the vicinity of the target protein region for DNA cleavage. This process is limited by the binding ability of the PA / PG protein to the Fc region of the secondary antibody, and the non-specific binding of the antibody and chromatin accessibility can both affect the final data. Non-specifically binding antibodies can guide TN5 or MNase enzymes to cleave in non-target regions, leading to false positives. Furthermore, transcription factors (TFs) primarily bind to the nucleosome-depleted region (NDR) in open chromatin regions (Maxime M., et al. Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor. Molecular Cell. 2019; 77:488-500). Figure 3 In CUT&Tag or CUT&Run experiments, abnormal chromatin state regulation can affect the detection of transcription factor targets and excitatory histone modification targets. Therefore, regulating the chromatin state in CUT&Tag and CUT&Run technologies and reducing non-specific antibody binding can effectively improve the detection capability of the technology and the final sequencing data quality. Summary of the Invention

[0005] The purpose of this application is to provide a composition and a method for constructing a gene library for studying protein-DNA interactions. By reducing the salt ion concentration in the transposase binding buffer, the detection effect on transcription factor targets and histone modification targets is improved, while reducing non-specific binding of antibodies and reducing false positives in CutTag and Cut Run experiments.

[0006] A first aspect of this application provides a composition comprising a buffering component, NaCl, KCl, spermidine, a cell permeabilizing agent, and a protease inhibitor, wherein the concentration of NaCl is 10-300 mM, for example 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM.

[0007] In some embodiments, the cell permeabilizing agent is, for example, Tween20, digitalis saponins, saponins, polyethylene glycol octylphenyl ether, ethylphenyl polyethylene glycol, etc.

[0008] In some embodiments, the buffering component is, for example, HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), Tris, Tris-HCl, MOPS, PBS, etc.

[0009] In some embodiments, the composition comprises HEPES, NaCl, KCl, spermidine, digitalis saponins, and a protease inhibitor, wherein the concentration of NaCl is 10-300 mM, for example 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM.

[0010] In some embodiments, the concentration of KCl is 50-300 mM, for example 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM or 300 mM.

[0011] In some embodiments, the concentration of HEPES is 10-100 mM, such as 10 mM, 12 mM, 15 mM, 18 mM, 20 mM, 22 mM, 25 mM, 28 mM, 30 mM, 35 mM, 40 mM, 45 mM and 50 mM.

[0012] In some embodiments, the concentration of spermidine is 0.1-5 mM, for example 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, or 2 mM.

[0013] In some embodiments, the concentration of the digitalis saponin (mass-volume ratio, 1% = 1 g / 100 ml) is 0.01%-0.1%, for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%. In some embodiments, the digitalis saponin is present in the composition in an amount sufficient to maintain cell permeability and allow primary antibodies, secondary antibodies, and transposons to enter the cells.

[0014] In some embodiments, the protease inhibitor has broad-spectrum protease inhibitory activity, including but not limited to commercially available or non-commercial protease inhibitors known in the art.

[0015] In some embodiments, the amount of the protease inhibitor is added according to the amount specified in the respective product instructions or an amount known in the art.

[0016] In some embodiments, the composition comprises 10-50 mM HEPES, 50-300 mM NaCl, 50-300 mM KCl, 0.1-5 mM spermidine, 0.01%-0.1% digitalis saponins, and an effective amount of protease inhibitor. In some embodiments, the composition comprises 10-30 mM HEPES, 50-300 mM NaCl, 50-300 mM KCl, 0.1-2 mM spermidine, 0.01%-0.05% digitalis saponins, and an effective amount of protease inhibitor. In some embodiments, the composition comprises 10-30 mM HEPES, 50-250 mM NaCl, 50-250 mM KCl, 0.1-1 mM spermidine, 0.01%-0.05% digitalis saponins, and an effective amount of protease inhibitor. In some embodiments, the composition comprises 20 mM HEPES, 50-250 mM NaCl, 50-250 mM KCl, 0.5 mM spermidine, 0.01% digitalis saponins, and an effective amount of protease inhibitor.

[0017] "Effective amount of protease inhibitor" refers to a certain concentration range of protease inhibitors that can effectively protect proteins in the reaction system from hydrolysis. Refer to the dosage of Vazyme TD903.

[0018] In some embodiments, the concentration of each component of the composition is 2 to 50 times that of the above-mentioned component concentrations, for example, 2 times, 3 times, 4 times, 5 times, 6 times, 8 times, 10 times, 12 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times or 50 times.

[0019] A second aspect of this application provides a transposase incubation system comprising a transposase complex and the composition of the first aspect.

[0020] In some embodiments, the transposase complex is an X-transposase complex, where X is, for example, Protein A, Protein G, or Protein AG, and the transposase is, for example, Tn5 transposase, which embeds a partially double-stranded oligonucleotide containing a transposon terminal sequence and a tag sequence, the tag sequence being used for subsequent PCR amplification.

[0021] In some embodiments, the concentration of the transposase complex in the transposase incubation system is 0.01-0.08 μM, for example 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.07 μM or 0.08 μM.

[0022] In some embodiments, the transposase incubation system further includes a sample, which is permeabilized cells. In some embodiments, the number of cells is at least 100, 500, 1000, 2000, 5000, 8000, or 10000.

[0023] In some embodiments, the numerical values ​​or ranges of this application include an error of up to ±20%, up to ±10%, up to ±5%, up to ±4%, up to ±3%, up to ±2%, or up to ±1%, wherein “up to” includes the stated number.

[0024] A third aspect of this application provides a transposase reaction composition comprising the composition of the first aspect and a divalent metal salt.

[0025] In some embodiments, the divalent metal salt is, for example, Ca. 2+ Salt, Mg 2+ Salt, Zn 2+ Salt, Mn 2+ Salt, Co 2+ Salt or one or more of these; for example, MgCl2; the concentration of the divalent metal salt is, for example, 5-20 mM, such as 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM or 20 mM.

[0026] The fourth aspect of this application provides a transposase reaction system comprising the transposase incubation system of the second aspect and a divalent metal salt.

[0027] In some embodiments, the divalent metal salt is, for example, Ca. 2+ Salt, Mg 2+ Salt, Zn 2+ Salt, Mn 2+ Salt, Co 2+ Salt or one or more of these; for example, MgCl2; the concentration of the divalent metal salt is, for example, 5-20 mM, such as 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM or 20 mM.

[0028] In some embodiments, the numerical values ​​or ranges of this application include an error of up to ±20%, up to ±10%, up to ±5%, up to ±4%, up to ±3%, up to ±2%, or up to ±1%, wherein “up to” includes the stated number.

[0029] The fifth aspect of this application provides a method for constructing a gene library for studying protein-DNA interactions, the method comprising:

[0030] Step (1) Collect cell samples, permeabilize them, add target protein (e.g., transcription factor) binding primary antibody and secondary antibody for incubation, wherein the target protein binding primary antibody binds to the target protein, and the secondary antibody binds to the target protein binding primary antibody;

[0031] Step (2) Add the transposase complex to the composition of the first aspect and incubate;

[0032] Step (3) Add the transposase reaction composition of the third aspect to carry out a fragmentation reaction to obtain fragmented DNA; wherein the transposase complex contains a group or substance capable of binding to the secondary antibody, a transposase, and an oligonucleotide containing a transposase recognition core sequence (ME sequence) and a tag sequence; the substance capable of binding to the secondary antibody is, for example, Protein A, Protein G, or Protein AG.

[0033] Step (4) Optionally, purify the fragmented DNA;

[0034] Step (5) Ligate sequencing adapters to the DNA fragments;

[0035] Step (6) Optionally, the product after adapter ligation is purified and / or amplified.

[0036] In some implementations, step (1) further includes incubating the collected cell sample with activated magnetic beads.

[0037] In some implementations, the target protein is a transcription factor or a histone.

[0038] In some embodiments, the activated magnetic beads are concanavalin A magnetic beads.

[0039] In some embodiments, the permeation includes adding an antibody incubation buffer containing a cell permeation reagent.

[0040] In some embodiments, the cell permeation agent is digitalis saponin or Tween, for example, 0.01%-0.1% digitalis saponin.

[0041] In some implementations, the cell sample is incubated with a primary antibody after binding to the target protein, and then incubated with a secondary antibody.

[0042] In some implementations, step (1) further includes separating and collecting the magnetic beads after the cell sample has been incubated with the activated magnetic beads, and / or separating and collecting the magnetic beads after the cells have been incubated with the target protein-binding primary and secondary antibodies.

[0043] In some implementations, step (2) further includes separating and collecting the magnetic beads after incubation is complete.

[0044] In some embodiments, the transposase is a Tn5 transposase, which is any Tn5 transposase known in the art or an active mutant thereof.

[0045] In some implementations, step (5) includes adding primers that match the tag sequence and have adapter sequences, and then amplifying the DNA fragment with the sequencing adapter by PCR.

[0046] In some embodiments, the primers include a first primer with a first adapter sequence and a second primer with a second adapter sequence.

[0047] In some implementations, the adapter sequence is a sequencing matrix-related sequence used by current or future sequencing platforms (including but not limited to the iontorrent platform, the Illumina platform, and the BGI platform).

[0048] In some implementations, the first connector sequence is a P5 connector sequence of the Illumina platform, the second connector sequence is a P7 connector sequence of the Illumina platform, and vice versa.

[0049] In some implementations, the first connector sequence is the P1 connector sequence of the ion torrent platform, and the second connector sequence is the A connector of the ion torrent platform, or vice versa.

[0050] In some implementations, the first connector sequence is an upstream clamping sequence for single-ended tag database creation on the MGI platform, and the second connector sequence is a downstream clamping sequence for single-ended tag database creation on the MGI platform, and vice versa. In some implementations, the first connector sequence is an upstream clamping sequence for double-ended tag database creation on the MGI platform, and the second connector sequence is a downstream clamping sequence for double-ended tag database creation on the MGI platform, and vice versa.

[0051] In some embodiments, the primer further includes an index sequence. In some embodiments, the index sequence is, for example, 6-8 bases, preferably 8.

[0052] In some implementations, the first primer is an N5 primer and the second primer is an N7 primer, or vice versa.

[0053] In some embodiments, the incubation step in step (2) includes incubation at room temperature for 0.5-2 hours, preferably incubation at room temperature for 1-2 hours, and more preferably rotational incubation at room temperature for 1-2 hours.

[0054] In some embodiments, the fragmentation reaction in step (3) includes incubation at 35-40°C for 0.5-2 hours, preferably at 35-38°C for 1-2 hours, and more preferably at 37°C for 1-2 hours.

[0055] In some embodiments, the method further includes a step of terminating the transposase fragmentation reaction, such as inactivating the Tn5 transposase, for example by adding EDTA, SDS or proteinase K, preferably 10% SDS.

[0056] In some embodiments, the purification step employs magnetic bead extraction, high-salt precipitation, centrifugal column extraction, or phenol-chloroform extraction, with magnetic bead extraction being preferred.

[0057] A sixth aspect of this application provides an antibody incubation composition comprising a buffering component, a metal salt, spermidine, a chelating agent, a stabilizer, a cell permeabilizing agent, and at least one nonionic surfactant.

[0058] In some embodiments, the cell permeabilizing agent is, for example, Tween 20, digitalis saponins, saponins, polyethylene glycol octylphenyl ether, ethylphenyl polyethylene glycol, etc.

[0059] In some embodiments, the buffering component is, for example, HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), Tris, Tris-HCl, MOPS, PBS, etc.

[0060] In some embodiments, the metal salt is, for example, a monovalent metal salt, such as Na. + Salt and K + Salts, such as NaCl.

[0061] In some embodiments, the chelating agent is, for example, ethylenediaminetetraacetic acid (EDTA), aminotriacetic acid (NTA), diethylenetriaminepentaacetic acid, hydroxyethylethylenediaminetriacetic acid (HEDTA), dihydroxyethylglycine (DEG), citric acid (CA), tartaric acid (TA), and gluconic acid (GA).

[0062] In some embodiments, the stabilizer is, for example, an enzyme stabilizer, such as BSA (bovine serum albumin).

[0063] In some embodiments, the nonionic surfactant is, for example, polysorbate-20 (Tween20), polysorbate-80 (Tween80), polyethylene glycol 4000 (PEG4000), polyethylene glycol 8000 (PEG8000), cetearyl alcohol polyether-10 (AEO-10), or polyethylene glycol octylphenyl ether (TritonX-100).

[0064] In some embodiments, the buffer solution comprises HEPES, NaCl, spermidine, EDTA, BSA, digitalis saponins, protease inhibitors, and at least one nonionic surfactant.

[0065] In some embodiments, the buffer solution comprises HEPES, NaCl, spermidine, EDTA, BSA, digitalis saponins, protease inhibitors, and at least one nonionic surfactant selected from Triton X-100 and Tween 20.

[0066] In some embodiments, the concentration (volume fraction) of Triton X-100 is 0.01-5%, preferably 0.01-2%, for example 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, and 2%.

[0067] In some embodiments, the concentration (volume fraction) of the Tween20 is 0.1%-5%, preferably 0.1%-3%, for example 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.2%, 2.5%, 2.7%, 2.8%, and 3%.

[0068] In some embodiments, the concentration of HEPES is 10-100 mM, preferably 10-50 mM, such as 10 mM, 12 mM, 15 mM, 18 mM, 20 mM, 22 mM, 25 mM, 28 mM, 30 mM, 35 mM, 40 mM, 45 mM and 50 mM.

[0069] In some embodiments, the concentration of NaCl is 50-200 mM, preferably 100-200 mM, such as 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM and 200 mM.

[0070] In some embodiments, the concentration of spermidine is 0.1-5 mM, preferably 0.1-2 mM, for example 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM and 2 mM.

[0071] In some embodiments, the concentration of the BSA (mass fraction) is 0.01-1%, preferably 0.05-0.5%, for example 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5%.

[0072] In some embodiments, the concentration of EDTA is 1-10 mM, preferably 1-5 mM, such as 1 mM, 1.2 mM, 1.5 mM, 1.8 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM and 5 mM.

[0073] In some embodiments, the concentration of the digitalis saponin (mass-volume ratio, 1% = 1 g / 100 ml) is 0.01-2%, preferably 0.01-1%, for example 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, and 1%. In some embodiments, the digitalis saponin is present in the composition in an amount sufficient to maintain cell permeability and allow primary antibodies, secondary antibodies, and transposons to enter the cells.

[0074] In some embodiments, the protease inhibitor has broad-spectrum protease inhibitory activity, including but not limited to commercially available or non-commercial protease inhibitors known in the art.

[0075] In some embodiments, the amount of the protease inhibitor is added according to the amount specified in the respective product instructions or an amount known in the art.

[0076] In some embodiments, the antibody incubation composition comprises 10-50 mM HEPES, 100-200 mM NaCl, 0.1-2 mM spermidine, 1-5 mM EDTA, 0.05-0.5% BSA, 0.01-1% digitalis saponins, and an effective amount of protease inhibitors, Triton X-100 and Tween 20. In some embodiments, the antibody incubation composition comprises 20 mM HEPES, 150 mM NaCl, 0.5 mM spermidine, 2 mM EDTA, 0.1% BSA, 0.05% digitalis saponins, and an effective amount of protease inhibitors, Triton X-100 and Tween 20. In some embodiments, the antibody incubation composition comprises 20 mM HEPES, 150 mM NaCl, 0.5 mM spermidine, 2 mM EDTA, 0.1% BSA, 0.05% digitalis saponins, an effective amount of protease inhibitor, 0.01-5% Triton X-100, and 0.1-5% Tween 20. In some embodiments, the antibody incubation composition comprises 20 mM HEPES, 150 mM NaCl, 0.5 mM spermidine, 2 mM EDTA, 0.1% BSA, 0.05% digitalis saponins, an effective amount of protease inhibitor, 0.01-2% Triton X-100, and 0.1-3% Tween 20.

[0077] "Effective amount of protease inhibitor" refers to a certain concentration range of protease inhibitors that can effectively protect proteins in the reaction system from hydrolysis.

[0078] In some embodiments, the concentration of each component of the antibody incubation composition is 2 to 50 times that of the above-mentioned component concentrations, for example, 2 times, 3 times, 4 times, 5 times, 6 times, 8 times, 10 times, 12 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times or 50 times.

[0079] A seventh aspect of this application provides an antibody incubation system comprising the antibody incubation composition of the sixth aspect and a target protein binding antibody.

[0080] In some embodiments, the target protein binding antibody is, for example, a target protein binding primary antibody and / or a secondary antibody. In some embodiments, the amount of the target protein binding primary antibody and / or secondary antibody is added according to the amount directed in the respective product instructions or amounts known in the art.

[0081] In some implementations, the target protein is a transcription factor or a histone.

[0082] In some embodiments, the antibody incubation system further includes a sample, which is permeabilized or non-permeabilized cells. In some embodiments, the number of cells is at least 100, 500, 1000, 2000, 5000, 8000, or 10000.

[0083] In some embodiments, the numerical values ​​or ranges of this application include an error of up to ±20%, up to ±10%, up to ±5%, up to ±4%, up to ±3%, up to ±2%, or up to ±1%, wherein “up to” includes the stated number.

[0084] The eighth aspect of this application provides a method for constructing a gene library for studying protein-DNA interactions, the method comprising:

[0085] Step (1) Collect cell samples, add target protein binding primary antibody and sixth aspect antibody incubation composition for incubation, permeate cells, the target protein binding primary antibody binds to target protein;

[0086] Step (2) Add secondary antibody and incubate, wherein the secondary antibody binds to the target protein and the primary antibody binds to it;

[0087] Step (3) Add the transposase complex and reaction buffer to perform a fragmentation reaction to obtain fragmented DNA; wherein the transposase complex contains a group or substance that can bind to the secondary antibody, a transposase, and an oligonucleotide containing a transposase recognition core sequence (ME sequence) and a tag sequence; the substance that can bind to the secondary antibody is, for example, Protein A, Protein G, or Protein AG.

[0088] Step (4) Optionally, purify the fragmented DNA;

[0089] Step (5) Ligate sequencing adapters to the DNA fragments;

[0090] Step (6) Optionally, the product after adapter ligation is purified and / or amplified.

[0091] In some implementations, the target protein is a transcription factor or a histone.

[0092] In some implementations, the incubation in step (1) includes incubation at 0-4°C for 6-24 hours, preferably at 0-4°C for 8-12 hours, and most preferably at 4°C for 8-12 hours.

[0093] In some implementations, step (1) further includes incubating the collected cell sample with activated magnetic beads.

[0094] In some embodiments, the activated magnetic beads are concanavalin A magnetic beads.

[0095] In some embodiments, step (1) further includes separating and collecting the magnetic beads after the cell sample has been incubated with the activated magnetic beads, and / or separating and collecting the magnetic beads after the cells have been incubated with the target protein-binding primary antibody.

[0096] In some implementations, step (2) further includes separating and collecting the magnetic beads after incubation is complete.

[0097] In some embodiments, the reaction buffer includes a transposase incubation buffer and a transposase reaction buffer.

[0098] In some embodiments, step (3) includes adding the transposase complex and transposase incubation buffer, incubating, adding transposase reaction buffer, and performing a fragmentation reaction.

[0099] In some embodiments, the transposase incubation buffer is, for example, the composition described in the first aspect of this application.

[0100] In some embodiments, the transposase reaction buffer is, for example, the transposase reaction composition described in the third aspect of this application.

[0101] In some embodiments, the transposase is a Tn5 transposase, which is any Tn5 transposase known in the art or an active mutant thereof.

[0102] In some implementations, step (5) includes adding primers that match the tag sequence and have adapter sequences, and then amplifying the DNA fragment with the sequencing adapter by PCR.

[0103] In some embodiments, the primers include a first primer with a first adapter sequence and a second primer with a second adapter sequence.

[0104] In some implementations, the adapter sequence is a sequencing matrix-related sequence used by current or future sequencing platforms (including but not limited to the iontorrent platform, the Illumina platform, and the BGI platform).

[0105] In some implementations, the first connector sequence is a P5 connector sequence of the Illumina platform, the second connector sequence is a P7 connector sequence of the Illumina platform, and vice versa.

[0106] In some implementations, the first connector sequence is the P1 connector sequence of the ion torrent platform, and the second connector sequence is the A connector of the ion torrent platform, or vice versa.

[0107] In some implementations, the first connector sequence is an upstream clamping sequence for single-ended tag database creation on the MGI platform, and the second connector sequence is a downstream clamping sequence for single-ended tag database creation on the MGI platform, and vice versa. In some implementations, the first connector sequence is an upstream clamping sequence for double-ended tag database creation on the MGI platform, and the second connector sequence is a downstream clamping sequence for double-ended tag database creation on the MGI platform, and vice versa.

[0108] In some embodiments, the primer further includes an index sequence. In some embodiments, the index sequence is, for example, 6-8 bases, preferably 8.

[0109] In some implementations, the first primer is an N5 primer and the second primer is an N7 primer, or vice versa.

[0110] In some embodiments, the incubation step in step (2) includes incubation at room temperature for 0.5-2 hours, preferably incubation at room temperature for 1-2 hours, and more preferably rotational incubation at room temperature for 1-2 hours.

[0111] In some embodiments, the fragmentation reaction in step (3) includes incubation at 35-40°C for 0.5-2 hours, preferably at 35-38°C for 1-2 hours, and more preferably at 37°C for 1-2 hours.

[0112] In some embodiments, the method further includes a step of terminating the transposase fragmentation reaction, such as inactivating the Tn5 transposase, for example by adding EDTA, SDS or proteinase K, preferably 10% SDS.

[0113] In some embodiments, the purification step employs magnetic bead extraction, high-salt precipitation, centrifugal column extraction, or phenol-chloroform extraction, with magnetic bead extraction being preferred.

[0114] The ninth aspect of this application provides a method for studying protein-DNA interactions, the method comprising: performing sequencing on a library constructed according to the fifth or eighth aspect of this invention.

[0115] The tenth aspect of this application provides a kit comprising at least one, two, or three of the compositions described in the first aspect of this application, the transposase reaction composition described in the third aspect, and the antibody incubation composition described in the sixth aspect.

[0116] In some embodiments, the kit is used to implement the methods provided in the fifth, eighth, and / or ninth aspects of the present invention.

[0117] In some implementations, the kit also includes other auxiliary detection reagents, such as transposase complexes, cell permeation reagents, purification magnetic beads, sequencing adapters, DNA polymerase, amplification buffer, amplification primers, and nuclease-free water.

[0118] This application can achieve the following beneficial effects:

[0119] 1. By modifying the salt concentration during transposase incubation, chromatin accessibility was improved, resulting in a significant increase in the proportion of small fragments in the library, indicating the recovery of more fragment signals from NDR regions (see appendix). Figure 4A-4 B).

[0120] 2. By modifying the salt concentration during transposase incubation, the final detection efficiency for transcription factor targets and excitatory histone modification targets was improved (see appendix). Figures 5A-5B Appendix Figures 6A-6B ).

[0121] 3. Modifying the antibody incubation buffer by adding additives such as Tween 20 and Triton X-100 significantly improved the TSS enrichment signal value, and the increased frip score indicates a significant improvement in the positive detection rate. (See attached image) Figures 7A-7B ). Attached Figure Description

[0122] Figure 1 This displays a schematic diagram of ChIP technology.

[0123] Figure 2 This displays a schematic diagram of the Cut & Tag technology.

[0124] Figure 3 This diagram shows that TF is mainly bound to the NDR area.

[0125] Figure 4A-1 , Figure 4A-2 , Figure 4A-3 , Figure 4A-4 and Figure 4A-5 The effects of transposase binding buffer (Dig-buffer) with different salt ion concentrations on the H3K4me3 target library preparation peak diagram are shown. Figure 4B This shows the effect of different salt ion concentrations of Dig-buffer on the proportion of the 180-350bp small fragment at the H3K4me3 target.

[0126] Figure 5A Heatmap plot showing library sequencing results for CTCF targets using Dig-buffer at different salt ion concentrations; Figure 5B The FRip score shows the sequencing results of libraries targeting CTCF targets using Dig-buffer with different salt ion concentrations.

[0127] Figure 6AHeatmap plot showing library sequencing results for the H3K4me3 target using Dig-buffer at different salt ion concentrations; Figure 6B The FRip score shows the sequencing results of libraries targeting H3K4me3 using Dig-buffer with different salt ion concentrations.

[0128] Figure 7A Heatmap plot showing the sequencing results of libraries targeting H3K4me3 using different antibody incubation buffers; Figure 7B The FRip score shows the sequencing results of libraries targeting H3K4me3 using different antibody incubation buffers. Detailed Implementation

[0129] The technical solution of this application will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of this application and do not represent or limit the scope of protection of this application. The scope of protection of this application shall be determined by the claims.

[0130] In the following embodiments, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the art; unless otherwise specified, all experimental methods and techniques used were conventional methods and techniques in the art.

[0131] Reagent preparation

[0132] Binding Buffer: Take 30 μL of 10× Binding Buffer (Vazyme#TD903), add ddH2O to 300 μL, and mix well.

[0133] 50× Protease Inhibitor: Dissolve one protease inhibitor mixed tablet (Sigma-Aldrich, 5056489001) in 1 mL ddH2O and store at -20℃.

[0134] Wash Buffer: Take 0.4 mL of 10×Wash Buffer (-) (HEPES 200 mM, NaCl 1.5 M, spermidine 5 mM), add 80 μL of 50× protease inhibitor, and add ddH2O to 4 mL and mix well.

[0135] Dig-wash Buffer: Take 2.97 mL of the Wash Buffer prepared above, add 30 μL of 5% Digitonin, and mix well.

[0136] Antibody Buffer 1 (Control): Mix 1 μL of 0.5 M EDTA, 0.8 μL of 30% BSA and 250 μL of Lig-wash Buffer.

[0137] Antibody Buffer 2, mix 1 μL 0.5M EDTA, 0.8 μL 30% BSA and 250 μL L ig-wash buffer, add Triton X-100 and Tween 20 to a final concentration of 0.05% and 0.5%.

[0138] Antibody Buffer 3: Mix 1 μL of 0.5M EDTA, 0.8 μL of 30% BSA and 250 μL of Ldiig-wash Buffer, add Triton X-100 and Tween 20 to a final concentration of 0.5% and 1%, cool on ice, and use immediately.

[0139] Antibody Buffer 4, mixed with 1 μL 0.5M EDTA, 0.8 μL 30% BSA and 250 μL L ig-wash buffer, and Triton X-100 and Tween 20 were added to a final concentration of 0.1% and 1.5%.

[0140] 10×Dig-400 Buffer(-): 200M HEPES, NaCl 4M, 5mM spermidine, KCl 4M.

[0141] 10×Dig-300 Buffer(-): 200M HEPES, NaCl 3M, 5mM spermidine, KCl 3M.

[0142] 10×Dig-250 Buffer(-): 200M HEPES, NaCl 2.5M, 5mM spermidine, KCl2.5M.

[0143] 10×Dig-150 Buffer(-): 200M HEPES, NaCl 1.5M, 5mM spermidine, KCl1.5M.

[0144] 10×Dig-50 Buffer(-): 200M HEPES, NaCl 500mM, 5mM spermidine, KCl 500mM.

[0145] 1×Dig-400 Buffer: Take 0.4 mL of 10×Dig-400 Buffer(-), add 8 μL of 5% Digitonin and 80 μL of 50× protease inhibitor, add ddH2O to 4 mL, and mix well.

[0146] 1×Dig-300 Buffer: Take 0.4 mL of 10×Dig-300 Buffer(-), add 8 μL of 5% Digitonin and 80 μL of 50× protease inhibitor, add ddH2O to 4 mL, and mix well.

[0147] 1×Dig-250 Buffer: Take 0.4 mL of 10×Dig-250 Buffer(-), add 8 μL of 5% Digitonin and 80 μL of 50× protease inhibitor, add ddH2O to 4 mL, and mix well.

[0148] 1×Dig-150 Buffer: Take 0.4 mL of 10×Dig-150 Buffer(-), add 8 μL of 5% Digitonin and 80 μL of 50× protease inhibitor, add ddH2O to 4 mL, and mix well.

[0149] 1×Dig-50 Buffer: Take 0.4 mL of 10×Dig-50 Buffer(-), add 8 μL of 5% Digitonin and 80 μL of 50× protease inhibitor, add ddH2O to 4 mL, and mix well.

[0150] Tagmentation Buffer 400: Take 300 μL of Dig-400 Buffer, add 3 μL of 1M MgCl2 and mix well. Prepare and use immediately.

[0151] Tagmentation Buffer 300: Take 300 μL of Dig-300 Buffer, add 3 μL of 1M MgCl2 and mix well. Prepare and use immediately.

[0152] Tagmentation Buffer 250: Take 300 μL of Dig-250 Buffer, add 3 μL of 1M MgCl2 and mix well. Prepare and use immediately.

[0153] Tagmentation Buffer 150: Take 300 μL of Dig-150 Buffer, add 3 μL of 1M MgCl2 and mix well. Prepare and use immediately.

[0154] Tagmentation Buffer 50: Take 300 μL of Dig-50 Buffer, add 3 μL of 1M MgCl2 and mix well. Prepare and use immediately.

[0155] Example 1

[0156] Using 10,000 HeLa cells (Haixing Biotechnology Co., Ltd., catalog number TCH-C193) as targets, a Cut & Tag experiment was performed to fragment the cell chromatin. The CTCF target antibody used was from Abcam (ab126778), and the H3K4me3 target antibody was from Abcam (ab8580). The specific steps are as follows:

[0157] (I) ConABeads (Vazyme#TD903) processing

[0158] 1. Take an 8-tube strip and add 100 μL of binding buffer to each sample.

[0159] 2. Use a pipette to fully resuspend the ConABeads, take 10 μL of ConABeads into the Binding Buffer from step 1, mix well, place on a magnetic rack, and discard the supernatant after the solution becomes clear.

[0160] 3. Remove the 8-tube strip from the magnetic rack, add 100 μL of Binding Buffer, and gently mix with a pipette.

[0161] 4. Place the 8-tube bundle on a magnetic rack. After the liquid has clarified, discard the supernatant and add 10 μL of Binding Buffer to resuspend the ConABeads.

[0162] (II) Cell Collection

[0163] 1. Collect and count cells at room temperature.

[0164] 2. Take the required number of cells into a 1.5 mL EP tube, centrifuge at 2,500 rpm (600 × g) for 5 min at room temperature, and discard the supernatant.

[0165] 3. Resuspend the cells in 500 μL Wash Buffer at room temperature, centrifuge at 2,500 rpm (600 × g) for 5 min, and discard the supernatant.

[0166] 4. Add 100 μL of Wash Buffer to each sample to resuspend the cells.

[0167] (III) Incubation of cells with ConABeads

[0168] 1. Transfer 100 μL of cells to an 8-tube containing activated ConABeads, invert to mix, and incubate at room temperature for 10 min, inverting to mix 2-3 times during the incubation period.

[0169] 2. Collect the reaction solution by instantaneous centrifugation (<100×g), place the 8-tube tube on a magnetic rack, and discard the supernatant after the solution becomes clear.

[0170] (iv) Antibiotic incubation

[0171] 1. Add 50 μL of pre-chilled Antibody Buffer 1 to each sample to resuspend the cell-magnetic bead complex.

[0172] 2. Add the antibody to the 8-tube at the concentration recommended in the antibody instructions (1:100) and mix by inverting the tube.

[0173] 3. Collect the liquid at the bottom of the tube by instantaneous centrifugation, and place the 8-tube set at 4°C overnight.

[0174] (V) Second Antibody Incubation

[0175] 1. Dilute the secondary antibody (abcam, ab6702) with Dig-wash Buffer at a volume ratio of 1:100 (1:100 dilution is generally recommended), 50 μL per sample.

[0176] 2. Take the 8-tube incubation of the primary antibody in step (IV), centrifuge briefly to collect the reaction solution, place the 8-tube on a magnetic rack, and discard the supernatant after the solution becomes clear.

[0177] 3. Add the diluted secondary antibody from step 1 of step (V) for incubation. Invert the container several times to mix the antibody with the cell-magnetic bead complex evenly. Incubate at room temperature for 30-60 minutes.

[0178] 4. Collect the reaction solution by instantaneous centrifugation, place the 8-tube tube on a magnetic rack, and discard the supernatant after the solution becomes clear.

[0179] 5. Add 200 μL of Dig-wash Buffer to the 8-tube strip and invert it several times to ensure that the Buffer is fully mixed with the cell-magnetic bead complex.

[0180] 6. Repeat steps 4-5 twice (3 times in total).

[0181] (vi) pA / G-Tnp (Vazyme#TD903) incubation

[0182] 1. Take 2 μL of pA / G-Tnp and add it to 98 μL of Dig Buffer for mixing (different experimental groups are incubated with different Dig Buffers, specifically Dig 400 Buffer - Dig 50 Buffer), with a final concentration of 0.04 μM.

[0183] 2. Take the 8-tube incubation of the secondary antibody in step (V), centrifuge briefly to collect the reaction solution, place the 8-tube on a magnetic rack, and discard the supernatant after the solution becomes clear.

[0184] 3. Add 100 μL of the pA / G-Tnp Pro transposon diluted in step 1 to each sample, and invert the sample several times to mix the transposon with the cell magnetic bead complex evenly.

[0185] 4. Incubate by rotation at room temperature for 1 hour.

[0186] 5. Perform instant centrifugation by placing the 8-tube tube on a magnetic rack and discarding the supernatant after the liquid has clarified.

[0187] 6. Add 200 μL of Dig-Buffer to the 8-tube (different experimental groups use different Dig Buffer for incubation, specifically Dig 400 Buffer to Dig 50 Buffer), and invert the tube several times to ensure that the Buffer and cell-magnetic bead complex are thoroughly mixed.

[0188] 7. Repeat steps 5-6 twice (3 times in total).

[0189] (vii) Fragmentation

[0190] 1. Take 50 μL of Tagmentation Buffer (different experimental groups use different Tagmentation Buffers for incubation, specifically Tagmentation Buffer 400-Tagmentation Buffer 50, corresponding to Dig 400Buffer-Dig50Buffer), mix thoroughly by blowing, and place on ice for later use.

[0191] 2. Take the 8-tube incubated in step (VI) pA / G-Tnp Pro, centrifuge briefly to collect the reaction liquid, place the 8-tube on a magnetic rack, and discard the supernatant after the liquid becomes clear.

[0192] 3. Add 50 μL of the Tagmentation Buffer prepared in step (VII) Fragmentation Step 1 to each sample (different experimental groups are incubated with different Tagmentation Buffers, specifically Tagmentation Buffer 400-Tagmentation Buffer 50), and mix well.

[0193] 4. Place the 8-tube strip in a PCR instrument and incubate at 37°C for 60 minutes.

[0194] 5. Centrifuge briefly, add 2 μL of 10% SDS, mix by inverting, and incubate at 55°C for 10 min, inverting 2-3 times during the incubation period.

[0195] 6. Perform a brief centrifugation, place the 8-tube set on a magnetic rack, let it stand for about 2-3 minutes, carefully transfer the supernatant to a new 8-tube set, and discard the magnetic beads.

[0196] (viii) DNA extraction (reagent from Vazyme#TD903)

[0197] 1. Add 5 μl Proteinase K, 100 μl Buffer L / B and 20 μl DNA Extract Beads to the fragmented sample, vortex thoroughly to mix, and incubate at 55°C for 10 min, inverting and mixing 2-3 times during the incubation period.

[0198] 2. Perform instant centrifugation. Place the 8-tube bundle on a magnetic rack and let it stand for about 2-3 minutes. Carefully remove the supernatant.

[0199] 3. Remove the above sample from the magnetic rack, add 200 μl of BufferWA (please confirm that anhydrous ethanol has been added before use), vortex thoroughly to mix, and collect the reaction solution by instant centrifugation. Place the 8-tube strip on the magnetic rack, let it stand for 2 minutes, and discard the supernatant.

[0200] 4. Remove the above sample from the magnetic rack, add 200 μl of BufferWB (please confirm that anhydrous ethanol has been added before use), vortex thoroughly to mix, and collect the reaction solution by instant centrifugation. Place the 8-tube strip on the magnetic rack, let it stand for 2 minutes, and discard the supernatant.

[0201] 5. Repeat step 4 once.

[0202] 6. Open the lid and let it air dry at room temperature for 5-10 minutes until there is no liquid residue in the tube and the surface of the magnetic beads is no longer reflective.

[0203] 7. Remove the above sample from the magnetic rack, add 22 μl of sterile ultrapure water, mix well by pipetting, and elute at room temperature for 5 min, gently shaking 2-3 times during the process.

[0204] 8. Place the 8-tube strip on a magnetic rack and wait for the solution to clarify (about 1 minute). Then, aspirate 20 μl of the supernatant into a new 8-tube strip. The sample can be stored at -30 to -15°C for a long time, avoiding repeated freeze-thaw cycles.

[0205] (ix) Library Expansion

[0206] Library amplification and purification: Take 15 μL of the DNA product from the previous stage and amplify the library using primers containing N5 / N7. Then, purify the amplified product using VAHTS DNA Clean Beads (Vazyme#N411) (see Vazyme TD903 steps 08-10 for details).

[0207] (x) The purified product was analyzed for peak shape using an Agilent 2100 Bioanalyzer, and the library was sequenced. The results are as follows: Figures 4A-1 to 4A-5 , Figure 4B , Figure 5A -B、 Figure 6A -B is shown.

[0208] Results analysis:

[0209] like Figure 4A-1 , Figure 4A-2 , Figure 4A-3 , Figure 4A-4 , Figure 4A-5 and Figure 4B As shown, using H3K4me3 as the target, modifying the concentrations of NaCl and KCl in the Dig Buffer can effectively change the distribution of library fragment sizes. Specifically, as the concentrations of NaCl and KCl decrease, the proportion of small fragments in the library increases (4%-27%), indicating that modifying the salt concentration can regulate chromatin accessibility, thereby obtaining more NDR (nucleosome depleted region) information.

[0210] like Figures 5A-5B As shown: High-throughput sequencing was performed on the libraries of each CTCF group, and TSS heatmap enrichment signal analysis was performed. The TSS heatmap shows that the detection results of transcription factor CTCF increased with decreasing NaCl and KCl concentrations, with the TSS heatmap signal increasing from 8 to 20, indicating more enriched positive signals. The FRiP Score refers to the proportion of specifically enriched reads to the total number of reads in the library. The higher the value, the better the enrichment and the more realistic the signal. With decreasing NaCl and KCl concentrations, the FRiP Score increased from 0.07 to 0.27.

[0211] like Figures 6A-6BAs shown: High-throughput sequencing was performed on individual libraries of H3K4me3, and TSS heatmap enrichment signal analysis was performed. The TSS heatmap shows that the detection result of the excitatory histone modification target H3K4me3 increases with decreasing NaCl and KCl concentrations. The TSS heatmap signal increased from 30 to 50, indicating more enriched positive signals. The FRiP Score refers to the proportion of specifically enriched reads to the total number of reads in the library. The higher the value, the better the enrichment and the more realistic the signal. With decreasing NaCl and KCl concentrations, the FRiP Score increased from 0.21 to 0.37.

[0212] Example 2

[0213] In Example 2, Cut & Tag experiments were performed using 10,000 HeLa cells (Haixing Biotechnology Co., Ltd., catalog number TCH-C193) as targets for transcription factor CTCF and excitatory histone modification target H3K4me3.

[0214] Dig-300 Buffer and Tagmentation Buffer 300 were used for the experiment. Except for the antibody buffer, the other operations were consistent with Example 1. Different experimental groups were incubated with different Antibody Buffers 1-4. The experimental results are as follows: Figures 7A-7B As shown.

[0215] Results analysis:

[0216] like Figures 7A-7B As shown: High-throughput sequencing was performed on the libraries of each H3K4me3 experimental group, and TSS heatmap enrichment signal analysis was performed. The TSS heatmap shows that the detection results of the excitatory histone modification target H3K4me3 increased with the increase of Tween 20 and Triton X-100 concentrations. The TSS heatmap singularity increased from 40 to 80, indicating that more positive signals were enriched. The FRiP Score refers to the proportion of specifically enriched reads to the total number of reads in the library. The higher the value, the better the enrichment and the more realistic the signal. With the addition and increase of Tween 20 and Triton X-100 concentrations, the FRiP Score increased from 0.27 to 0.45.

Claims

1. A method for constructing a gene library for studying protein-DNA interactions, the method comprising the following steps: (1) Collect cell samples, add target protein binding primary antibody and antibody incubation composition for incubation, permeate cells, the primary antibody binds to target protein, the antibody incubation composition contains buffer component, NaCl, spermidine, chelating agent, stabilizer, cell permeation agent, and at least one nonionic surfactant, the concentration of NaCl is 50-200mM; (2) Add secondary antibody for incubation, wherein the secondary antibody binds to the primary antibody; (3) A transposase complex is added to a reaction buffer containing buffer components, NaCl, KCl, spermidine, cell permeabilizer, protease inhibitor, and divalent metal salt, wherein the concentration of NaCl is 50-200 mM and the concentration of KCl is 50-200 mM, and a fragmentation reaction is performed to obtain fragmented DNA; wherein the transposase complex contains a group or substance that can bind to the secondary antibody, a transposase, and an oligonucleotide containing a transposase-recognized core sequence and a tag sequence; the substance that can bind to the secondary antibody is Protein A, Protein G, or Protein AG; (4) Purify fragmented DNA; (5) Ligate sequencing adapters to DNA fragments; (6) Purify and / or amplify the product after the adapter is ligated.

2. The method according to claim 1, wherein the buffering component is HEPES and the cell permeabilizing agent is digitalis saponin.

3. The method according to claim 1, wherein the chelating agent is EDTA, the stabilizer is BSA, and the nonionic surfactant is at least one of Triton X-100 and Tween 20.

4. The method according to claim 1, wherein the antibody incubation composition in step (1) comprises 10-50 mM HEPES, 50-200 mM NaCl, 0.1-5 mM spermidine, 1-5 mM EDTA, 0.05-0.5% BSA, 0.01-1% digitalis saponin by weight / volume, an effective amount of protease inhibitor, 0.01-5% Triton X-100 and 0.1-5% Tween 20, and the reaction buffer in step (3) comprises 10-50 mM HEPES, 50-200 mM NaCl, 50-200 mM KCl, 0.1-5 mM spermidine, 0.01%-0.1% digitalis saponin by weight / volume, an effective amount of protease inhibitor and divalent metal salt.

5. The method according to claim 1, wherein the antibody incubation composition in step (1) comprises 10-30 mM HEPES, 50-100 mM NaCl, 0.1-1 mM spermidine, 1-5 mM EDTA, 0.05-0.5% BSA, 0.01-1% digitalis saponin by weight / volume, an effective amount of protease inhibitor, 0.01-5% Triton X-100 and 0.1-5% Tween 20, and the reaction buffer in step (3) comprises 10-30 mM HEPES, 50-100 mM NaCl, 50-100 mM KCl, 0.1-1 mM spermidine, 0.01%-0.05% digitalis saponin by weight / volume, an effective amount of protease inhibitor and divalent metal salt.

6. The method according to any one of claims 1-5, wherein the divalent metal salt is Mg. 2+ Salt.

7. The method according to claim 1, wherein the transposase is a Tn5 transposase, and step (3) includes adding the transposase complex and transposase incubation buffer, incubating, adding transposase reaction buffer, and performing a fragmentation reaction.

8. A method for studying protein-DNA interactions, the method comprising: (1) Constructing a library using the method described in any one of claims 1-7; (2) Sequencing on the machine.

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