Method for researching nucleic acid-protein interaction

Abstract

The invention provides a method for researching nucleic acid-protein interaction, and belongs to the technical field of biology. Specifically, by improving the formula of a reaction system and adopting a scheme of improving the permeability of a cell membrane or a nuclear membrane and reducing the release temperature of a nucleic acid fragment, the signal-to-noise ratio, the sensitivity and the resolution of an experimental data result are improved, and the detection effect of protein-nucleic acid interaction research is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for studying nucleic acid-protein interaction. BACKGROUND

[0002] Epigenetics and epitranscriptomics are key regulatory mechanisms that shape gene expression and protein translation. Epigenetic regulation refers to the process of regulating the content and function of nucleic acids or proteins in cells through epigenetic modifications (such as DNA methylation, modification of histone proteins in chromatin, and regulation of non-coding RNA, etc.) without changing the DNA sequence. All of these processes have one thing in common, which is to adjust the chromatin state to regulate gene expression. In eukaryotic genomes, nucleosomes are the basic structural units of chromatin, composed of DNA wrapped around four core histones. Regions with less nucleosome distribution are conducive to the binding and interaction of transcription factors and other regulatory elements with promoters and enhancers, thereby regulating gene expression. Regions with dense nucleosome structure are relatively closed to the structural regions related to gene expression. Disruption of gene expression controlled by epigenetics can lead to various diseases, therefore, studying protein-nucleic acid interaction patterns is of great significance for subsequent disease research.

[0003] Affinity-based methods for studying protein-DNA interactions mainly capture target proteins or DNA to study their interactions. These methods usually rely on antibodies, tags or other affinity reagents to enrich target molecules, such as ChIP, CUT&Tag, CUT&RUN, etc.

[0004] CUT&RUN technology was first developed and published by the Steven Henikoff laboratory in 2017. Its design inspiration comes from early chromatin cleavage techniques (such as ChIC), combined with the specific cleavage ability of micrococcal nuclease (MNase). The core of CUT&RUN technology is: direct targeted cleavage in the cell nucleus, avoiding the background noise caused by chromatin cross-linking and fragmentation in ChIP, using MNase to cleave DNA near the target protein binding site, releasing protein-DNA complexes outside the cell / nucleus. Nucleic acid extraction and nucleic acid sequencing library construction can be performed on the released products to obtain nucleic acid-protein interaction binding maps; protein mass spectrometry can also be performed to obtain proteomics data. Compared with ChIP-seq technology, CUT&RUN technology has a higher signal-to-noise ratio. However, compared with CUT&Tag, CUT&RUN technology has lower signal-to-noise ratio, lower sensitivity, and poor experimental stability, which limits the application of CUT&RUN technology, and its application range is much lower than that of CUT&Tag.

[0005] Therefore, there is an urgent need in the art to develop methods that can improve the performance of CUT&RUN technology. SUMMARY

[0006] The purpose of the present application is to provide a new method for targeted cleavage of nucleic acid by nucleases. The present application uses a method to improve the permeability of cell membranes (or nuclear membranes) and reduce the temperature at which nucleic acid fragments are released. This method solves the problems of low signal-to-noise ratio, low resolution, and low sensitivity in traditional affinity-targeted nuclease methods for studying nucleic acid-protein interactions, and improves the application effect of CUT&RUN and similar technologies.

[0007] In a first aspect of the present application, a method for constructing a nucleic acid library is provided, comprising: (S1) providing a cell or cell nucleus sample; (S2) washing or resuspending the cell or cell nucleus sample to obtain a cell or cell nucleus suspension; (S3) pretreating the cell or cell nucleus suspension to obtain a cell or cell nucleus precipitate; (S4) treating the cell or cell nucleus precipitate with an antibody to obtain an antibody-cell or cell nucleus complex; (S5) treating the antibody-cell or cell nucleus complex with a nuclease to obtain a nuclease-antibody-cell or cell nucleus complex; (S6) treating the nuclease-antibody-cell or cell nucleus complex with a protein-nucleic acid fragment complex release agent (hereinafter referred to as a release agent) in a metal ion-containing buffer at a temperature T (°C) for t minutes (min) in an activation release reaction system to fragment and release nucleic acid fragments, wherein the temperature T is 0-30°C, preferably 4-20°C, and more preferably 18°C; the treatment time t is 20-240 min, preferably 40-90 min, and more preferably 60 min; (S7) extracting the released nucleic acid fragments to prepare a nucleic acid library.

[0008] In another preferred embodiment, the pretreatment method comprises using magnetic beads, centrifugation, or other methods to capture cells or cell nuclei.

[0009] In another preferred embodiment, the other method for capturing cells or cell nuclei comprises capturing cells or cell nuclei on a solid surface by coupling proteins.

[0010] In another preferred embodiment, the protein-nucleic acid fragment complex release agent is selected from the group consisting of non-ionic surfactants, ionic surfactants, biologically active perforators, polar solvents, or combinations thereof.

[0011] In another preferred embodiment, the non-ionic surfactant is selected from the group consisting of polysorbate series compounds, alkylphenol polyoxyethylene ether compounds, NP40 or IGEPAL CA-630, digitonin, or a combination thereof.

[0012] In another preferred embodiment, the polysorbate series compounds are selected from the group consisting of Tween-20, Tween-21, Tween-40, Tween-60, Tween-61, Tween-65, Tween-80, Tween-81, Tween-85, or a combination thereof.

[0013] In another preferred embodiment, the alkylphenol polyoxyethylene ether compounds are selected from the group consisting of Triton X-100, Triton X-114, Triton X-15, Triton X-35, Triton X-45, or a combination thereof.

[0014] In another preferred embodiment, the ionic surfactant is selected from the group consisting of CHAPS, sodium deoxycholate, or a combination thereof.

[0015] In another preferred embodiment, the biologically active perforator is selected from the group consisting of streptolysin O, melittin, or a combination thereof.

[0016] In another preferred embodiment, the polar solvent is selected from the group consisting of DMSO, DMF, or a combination thereof.

[0017] In another preferred embodiment, the antibody comprises a primary antibody, or a combination of a primary antibody and a secondary antibody. The secondary antibody incubation can be performed as needed to enhance the primary antibody signal.

[0018] In another preferred embodiment, the concentration of the protein nucleic acid fragment complex releasing agent is 0.01% - 50% (v / v), preferably 0.05% - 25% (v / v).

[0019] In another preferred embodiment, the concentration of Tween 20 is 0.01% - 5% (v / v), preferably 0.05% - 0.1% (v / v).

[0020] In another preferred embodiment, the concentration of NP40 is 0.01% - 5% (v / v), preferably 0.05% - 0.1% (v / v).

[0021] In another preferred embodiment, the concentration of IGEPAL CA-630 is 0.01% - 5% (v / v), preferably 0.05% - 0.1% (v / v).

[0022] In another preferred embodiment, the concentration of CHAPS is 0.01% - 5% (v / v), preferably 0.05% - 0.1% (v / v).

[0023] In another preferred embodiment, the concentration of Triton X-100 is 0.01% - 5 %(v / v), preferably 0.05% - 0.1 %(v / v).

[0024] In another preferred embodiment, the concentration of sodium deoxycholate is 0.01% - 5 %(v / v), preferably 0.05% - 0.1 %(v / v).

[0025] In another preferred embodiment, the concentration of digitonin is 0.01% - 5 %(v / v), preferably 0.05% - 0.1 %(v / v).

[0026] In another preferred embodiment, the volume percentage of DMSO is 0.5% - 50 %(v / v), preferably 2.5% - 25 %(v / v).

[0027] In another preferred embodiment, the concentration of streptolysin O is 0.01 - 20 μg / mL.

[0028] In another preferred embodiment, the concentration of melittin is 0.1 - 500 μM.

[0029] In another preferred embodiment, the volume percentage of DMF is 0.5% - 50 %(v / v), preferably 2.5% - 25 %(v / v).

[0030] In another preferred embodiment, in step (S6), the temperature T is 0 ~ 30 ℃, preferably 4 ~ 20 ℃.

[0031] In another preferred embodiment, in step (S6), the time t is 20 ~ 240 min, preferably 40 ~ 90 min.

[0032] In another preferred embodiment, in step (S6), the temperature T in the process of fragmentation of the nucleic acid is a fixed temperature value or a combination of multiple temperature values; when the temperature is a combination of multiple temperature values, the process includes reactions at different temperatures respectively.

[0033] In another preferred embodiment, in step (S6), the temperature T in the process of activation and release can be a certain fixed value or a combination of multiple temperature values. For example, T is a fixed value of 18 ℃, that is, the temperature of activation and release is 18 ℃ throughout the reaction, and the reaction lasts for one hour. T can also be a combination of multiple values, including 15 ℃, 18 ℃ and 20 ℃, that is, the temperature of activation and release is divided into multiple segments: 15 ℃ for 20 min, 18 ℃ for 20 min, and 20 ℃ for 20 min.

[0034] In another preferred embodiment, the process of the fragmentation treatment of the nucleic acid in step (S6) comprises: a first activation release reaction, a second activation release reaction, a third activation release reaction, or a combination thereof; wherein, the first activation release reaction has a temperature of 15℃ and a reaction time of 20 min; the second activation release reaction has a temperature of 18℃ and a reaction time of 20 min; the third activation release reaction has a temperature of 20℃ and a reaction time of 20 min.

[0035] In another preferred embodiment, the metal chelator is selected from the group consisting of EDTA, EGTA, or a combination thereof.

[0036] In another preferred embodiment, the concentration of the metal ion chelator is 1-50 mM.

[0037] In another preferred embodiment, the metal chelator comprises EDTA at a concentration of 1-50 mM and EGTA at a concentration of 1-5 mM.

[0038] In another preferred embodiment, step (S6) comprises any one step selected from the group consisting of: (S61) performing the fragmentation treatment of the nucleic acid and releasing the nucleic acid fragments by treating the nuclease-antibody-cell or cell nucleus complex in an activation release reaction system at a temperature T1 (℃) for a time t1 (min) using a protein nucleic acid fragment complex release agent; (S62) performing the fragmentation treatment of the nucleic acid and releasing the nucleic acid fragments by treating the nuclease-antibody-cell complex in an activation release reaction system at a temperature T2 (℃) for a time t2 (min) using a protein nucleic acid fragment complex release agent; adding a metal chelator, treating at a temperature T3 (℃) for a time t3 (min), stopping the reaction and further releasing the nucleic acid fragments; (S63) treating the nuclease-antibody-cell complex in a metal ion buffer (without a protein nucleic acid fragment complex release agent) at a temperature T4 (℃) for a time t4 (min), and subsequently performing the reaction procedure of step S61; (S64) treating the nuclease-antibody-cell complex in a metal ion buffer (without a protein nucleic acid fragment complex release agent) at a temperature T4 (℃) for a time t4 (min), and subsequently performing the reaction procedure of step S62.

[0039] In another preferred embodiment, the temperature T1 is 0-30℃, preferably 10-20℃.

[0040] In another preferred embodiment, the time t1 is 20-240 min, preferably 40-60 min.

[0041] In another preferred embodiment, the temperature T2 is 0-30 ℃, preferably 4-10 ℃.

[0042] In another preferred embodiment, the time t2 is 20-240 min, preferably 20-40 min.

[0043] In another preferred embodiment, the temperature T3 is 0-30 ℃, preferably 10-20 ℃.

[0044] In another preferred embodiment, the time t3 is 20-240 min, preferably 20-50 min.

[0045] In another preferred embodiment, the temperature T4 is 0-30 ℃, preferably 0-10 ℃.

[0046] In another preferred embodiment, the time t4 is 1-240 min, preferably 20-60 min.

[0047] In another preferred embodiment, the temperature T1-T4 can be a single temperature throughout or a combination of multiple temperatures in stages.

[0048] In another preferred embodiment, in step (S6), the activated release reaction system comprises a protein nucleic acid fragment complex release agent and a metal ion buffer with a concentration of 0.01 mM-10 mM.

[0049] In another preferred embodiment, the metal ion in the metal ion buffer is selected from the group consisting of calcium ion, magnesium ion, manganese ion, iron ion, zinc ion, cobalt ion, or a combination thereof.

[0050] In another preferred embodiment, the concentration of the metal ion in the metal ion buffer is 0.01 mM-10 mM.

[0051] In another preferred embodiment, the metal ion buffer comprises a CaCl2 solution with a concentration of 0.01 mM-10 mM.

[0052] In another preferred embodiment, in step (S6), the activated release reaction system comprises digitalis saponin with a volume percentage of 0.01%-0.5 %(v / v), DMSO with a volume percentage of 0.5%-50 %(v / v), and a CaCl2 solution with a concentration of 0.01 mM-10 mM.

[0053] In another preferred embodiment, in step (S6), the activated release reaction system has a pH of 6.0-8.5.

[0054] In another preferred embodiment, in step (S6), the activation release reaction system further comprises a permeation buffer.

[0055] In another preferred embodiment, the permeation buffer comprises PB, Tris and HEPES, or a combination thereof.

[0056] In another preferred embodiment, the permeation buffer has a pH value of 7.0-7.8; preferably 7.2-7.6.

[0057] In another preferred embodiment, the permeation buffer comprises 1-500 mM NaCl or other salt with equivalent conductivity.

[0058] In another preferred embodiment, in (S1), the cell comprises a eukaryotic cell, preferably an animal or plant cell, more preferably a mammalian cell.

[0059] In another preferred embodiment, the mammalian cell is selected from the group consisting of a hamster cell, a human cell or a mouse cell.

[0060] In another preferred embodiment, in (S1), the cell sample has a cell density of 1 x 10 3 ~5 x 10 6 cells / mL.

[0061] In another preferred embodiment, in (S1), the cell sample is a cell nucleus sample obtained by treating cells having a cell density of 1 x 10 3 ~5 x 10 6 cells / mL with a cell lysis solution.

[0062] In another preferred embodiment, in (S2), the washing comprises adding a cell washing buffer, centrifuging to remove the supernatant to obtain a cell or cell nucleus sample, and resuspending the cell or cell nucleus sample with the cell washing buffer.

[0063] In another preferred embodiment, the cell washing buffer comprises HEPES-KOH at a final concentration of 10-30 mM, NaCl at a final concentration of 100-300 mM, spermidine at a final concentration of 0.1-5 mM, and water.

[0064] In another preferred embodiment, the magnetic beads are Concanavalin A magnetic beads, hereinafter referred to as ConA magnetic beads.

[0065] In another preferred embodiment, the pre-treatment of the pre-treated magnetic beads comprises the following steps: treating a ConA magnetic bead solution with a binding buffer, placing the solution on a magnetic stand to stand until the solution is clear, discarding the supernatant, and obtaining the pre-treated magnetic beads.

[0066] In another preferred embodiment, in step (S4), the incubation time of the antibody is 10 minutes to 20 hours.

[0067] In another preferred embodiment, step (S4) further comprises binding the antibody with a secondary antibody.

[0068] In another preferred embodiment, in step (S5), the nuclease is selected from the group consisting of pAG-DNase I, pAG-MNase, pAG-Benzonase, or a combination thereof.

[0069] In another preferred embodiment, the DNase I in the pAG-DNase I is cloned from bovine (Bos taurus). Bos taurus ).

[0070] In another preferred embodiment, the MNase in the pAG-MNase is cloned from Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus ).

[0071] In another preferred embodiment, the Benzonase in the pAG-Benzonase is cloned from Serratia marcescens (Serratia marcescens). Serratia marcescens ).

[0072] In another preferred embodiment, in step (S5), the nuclease is fused with a domain or polypeptide capable of binding to an antibody, so as to target the nuclease to be fixed near the target protein.

[0073] In another preferred embodiment, the domain or polypeptide capable of binding to an antibody is selected from the group consisting of protein A, protein G, protein L, or a combination thereof.

[0074] In another preferred embodiment, the domain or polypeptide capable of binding to an antibody is selected from an antibody capable of specifically recognizing the FC region of an antibody; the nuclease is targeted and fixed near the target protein by the antibody, so as to cut the nucleic acid to form a free nucleic acid-protein complex.

[0075] In another preferred embodiment, the nuclease has endonuclease activity, and is capable of cutting single-stranded or double-stranded nucleic acid without depending on specific sequences, and has a metal ion activation feature.

[0076] In another preferred embodiment, the nuclease includes but is not limited to pAG-DNase I, pAG-MNase, or pAG-Benzonase.

[0077] In another preferred embodiment, the nuclease has a metal ion activation feature.

[0078] In another preferred embodiment, step (S5) further comprises the following step: after the treatment, the nuclease is washed with a detergent, and the washing time is 1 to 60 minutes.

[0079] In another preferred embodiment, in step (S7), the extracting comprises: centrifuging the released nucleic acid fragments to obtain a supernatant, and adding a DNA extraction reagent to the supernatant for incubation.

[0080] In another preferred embodiment, in step (S7), the DNA extraction reagent comprises: 2-10 μL of 0.5 M EDTA; 1-8 μL of 10% SDS; and 0.2-5 μL of 20 mg / mL of proteinase K.

[0081] In the second aspect of the present application, a kit for constructing a nucleic acid library by the method of the first aspect of the present application is provided, comprising: (1) a protein nucleic acid fragment complex releasing agent; (2) a metal ion buffer; (3) a cell washing buffer; and (4) a fragmentation reaction reagent.

[0082] In another preferred embodiment, the protein nucleic acid fragment complex releasing agent is selected from the group consisting of a non-ionic surfactant, an ionic surfactant, a bioactive perforator, a polar solvent, or a combination thereof.

[0083] In another preferred embodiment, the non-ionic surfactant is selected from the group consisting of a polysorbate series compound, an alkyl phenol polyoxyethylene ether compound, NP40 or IGEPAL CA-630, a digitalis saponin, or a combination thereof.

[0084] In another preferred embodiment, the polysorbate series compound is selected from the group consisting of Tween-20, Tween-21, Tween-40, Tween-60, Tween-61, Tween-65, Tween-80, Tween-81, Tween-85, or a combination thereof.

[0085] In another preferred embodiment, the alkyl phenol polyoxyethylene ether compound is selected from the group consisting of Triton X-100, Triton X-114, Triton X-15, Triton X-35, Triton X-45, or a combination thereof.

[0086] In another preferred embodiment, the ionic surfactant is selected from the group consisting of CHAPS, sodium deoxycholate, or a combination thereof.

[0087] In another preferred embodiment, the bioactive perforator is selected from the group consisting of streptolysin O, melittin, or a combination thereof.

[0088] In another preferred embodiment, the polar solvent is selected from the group consisting of DMSO, DMF, or a combination thereof.

[0089] In another preferred embodiment, the protein nucleic acid fragment complex releasing agent is selected from the group consisting of DMSO, SMF, Tween-20, NP40, Triton X-100, CHAPS, sodium deoxycholate, streptolysin O, melittin, or a combination thereof.

[0090] In another preferred embodiment, the volume percentage of DMSO is 0.5%-50% (v / v), preferably 2.5%-25% (v / v).

[0091] In another preferred embodiment, the concentration of streptolysin O is 0.01-20 μg / mL.

[0092] In another preferred embodiment, the concentration of melittin is 0.1-500 μM.

[0093] In another preferred embodiment, the volume percentage of DMF is 0.5%-50% (v / v), preferably 2.5%-25% (v / v).

[0094] In another preferred embodiment, the metal ion in the metal ion buffer is selected from the group consisting of calcium ion, magnesium ion, manganese ion, iron ion, zinc ion, cobalt ion, or a combination thereof.

[0095] In another preferred embodiment, the concentration of the metal ion in the metal ion buffer is 0.01 mM-10 mM.

[0096] In another preferred embodiment, the metal ion buffer comprises a CaCl2 solution with a concentration of 0.01 mM-10 mM.

[0097] In another preferred embodiment, the kit further comprises a metal chelator.

[0098] In another preferred embodiment, the metal chelator is selected from the group consisting of EDTA, EGTA, or a combination thereof.

[0099] In another preferred embodiment, the concentration of the metal ion chelator is 1-50 mM.

[0100] In another preferred embodiment, the metal chelator comprises EDTA with a concentration of 1-50 mM and EGTA with a concentration of 1-5 mM.

[0101] In another preferred embodiment, the kit further comprises a permeation buffer.

[0102] In another preferred embodiment, the permeation buffer comprises PB, Tris, HEPES buffer.

[0103] In another preferred embodiment, the pH value of the permeation buffer is 7.0-7.8; preferably 7.2-7.6.

[0104] In another preferred embodiment, the permeation buffer further comprises 50-150 mM NaCl or other salt with equivalent conductance.

[0105] In another preferred embodiment, the permeation buffer further comprises a cell protein protective agent, which is 1-100 mM Glycine, 0.1%-2% (v / v) BSA, or other similar agent.

[0106] In another preferred embodiment, the permeation buffer comprises a detergent selected from the group consisting of: digitonin, Tween 20, SDS, NP40, CHAPS, sodium deoxycholate, or a combination thereof; preferably digitonin, Tween 20, SDS or NP40.

[0107] In another preferred embodiment, the cell washing buffer has a pH value of 7.2-7.6.

[0108] In another preferred embodiment, the cell washing buffer comprises 100-150 mM NaCl or other salt with equivalent conductance.

[0109] In another preferred embodiment, the fragmentation reagent includes, but is not limited to, a nuclease, a magnetic bead, a primary antibody, a secondary antibody.

[0110] In another preferred embodiment, the kit further comprises a DNA extraction reagent, a reaction termination reagent, or a reagent for PCR amplification.

[0111] In another preferred embodiment, the DNA extraction reagent comprises: EDTA, SDS, proteinase K.

[0112] In a third aspect of the present application, a method for measuring protein-nucleic acid interaction is provided, comprising: (S1) constructing a nucleic acid library using the method as described in the first aspect of the present application; and (S2) sequencing the nucleic acid library to generate a plurality of sequencing reads for measuring protein-nucleic acid interaction.

[0113] In another preferred embodiment, the protein comprises a DNA-binding protein with weak DNA-binding force or high dynamicity of DNA binding.

[0114] It should be understood that, within the scope of the present application, each of the technical features described above and in the following (e.g., in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0115] Figure 1The figure shows the comparison between the traditional CUT&RUN experimental process and the CUT&RUN LT process of the application.

[0116] Figure 2 The figure shows the IGV peak plot of the Pol-II antibody group with or without DMSO added, wherein the upper and lower figures represent the IGV peak plot with a genome coordinate scale of 50 kb and 20 kb, respectively.

[0117] Figure 3 The figure shows the IGV peak plot of the CTCF antibody group with or without DMSO added, and the bottom row is the control peak plot of ChIP-seq, wherein the genome coordinate scale is 50 kb.

[0118] Figure 4 The figure shows the IGV peak plot of the H3K4me3 antibody group with or without DMSO added, and the bottom row is the control peak plot of ChIP-seq, wherein the genome coordinate scale is 50 kb.

[0119] Figure 5 The figure shows the IGV peak plot of the Pol-II antibody with different DMSO groups at different activation temperatures, wherein the activation temperature in A is 4°C, and the activation temperature in B is 20°C.

[0120] Figure 6 The figure shows the IGV peak plot of the Pol-II antibody group at different activation temperatures.

[0121] Figure 7 The figure shows the IGV peak plot of the Pol-II antibody group using different CUT&RUN methods.

[0122] Figure 8 The figure shows the IGV peak plot of the CTCF antibody group using different CUT&RUN methods.

[0123] Figure 9 The figure shows the IGV peak plot of different antibody groups with different nuclease and different DMF added, wherein A is the Pol-II antibody group, and B is the CTCF antibody group.

[0124] Figure 10 The figure shows the IGV peak plot of the cell nucleus sample with or without DMSO added in different antibody groups, wherein A is the Pol-II antibody group, B is the CTCF antibody group, and C is the H3K4me3 antibody group, and the bottom row of B and C is the control peak plot of ChIP-seq.

[0125] Figure 11 The figure shows the IGV peak plot of the Pol-II antibody group with different concentrations of protein nucleic acid fragment complex releasing agent added.

[0126] Figure 12IGV peak plot of Pol-II antibody set after adding different concentrations of protein-promoting nucleic acid fragment complex release agent, wherein SLO is an abbreviation of streptolysin O, and MLT is an abbreviation of melittin.

[0127] Figure 13 IGV peak plot of Pol-II antibody set after adding different combinations of protein-promoting nucleic acid fragment complex release agent.

[0128] Figure 14 IGV peak plot of Pol-II antibody and CTCF antibody one-step multi-temperature gradient and protein-promoting nucleic acid fragment complex release agent in the activation system. DETAILED DESCRIPTION

[0129] The inventors have made an unexpected discovery through extensive research and a large number of experiments. By changing the enzyme cutting and nucleic acid release conditions of the traditional CUT&RUN (0-4℃ low-temperature enzyme cutting of chromatin DNA + 37℃ high-temperature release of enzyme-cut fragments), the inventors first discovered that the addition of a cell protein-promoting nucleic acid fragment complex release agent to the enzyme cutting system and the use of low-temperature nucleic acid fragment release can significantly improve the experimental quality, and the signal-to-noise ratio and sensitivity of the experimental results data are far superior to those of the traditional method. Some experimental results are even superior to those of the CUT&Tag technology, and the results of testing various nucleases are superior to those of the traditional CUT&RUN. On this basis, the present application was completed.

[0130] TERMS Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0131] As used herein, the terms “comprising” or “containing” include “including”, “consisting essentially of”, “consisting essentially of”, and “consisting of”; “consisting essentially of”, “consisting essentially of”, and “consisting of” are sub-concepts of “containing”, “having”, or “including”.

[0132] As used herein, “the CUT&RUNLT method of the present application”, “the CUT&RUNLT process of the present application”, “the CUT&RUNLT one-step method of the present application”, and “the CUT&RUNLT two-step method of the present application” can be used interchangeably, and all refer to the method of the first aspect of the present application, specifically, by changing the enzyme cutting and nucleic acid release conditions of the traditional CUT&RUN (0-4℃ low-temperature enzyme cutting of chromatin DNA + 37℃ high-temperature release of enzyme-cut fragments), adding a cell protein-promoting nucleic acid fragment complex release agent to the enzyme cutting system, and using a low-temperature nucleic acid fragment release method.

[0133] As used herein, "pro-nucleic acid fragment complex release agent", "cell permeabilizing agent" and "pro-release agent" are used interchangeably and refer to an agent capable of facilitating the release of a nucleic acid fragment or a complex formed by the nucleic acid fragment and an antibody in the methods described herein.

[0134] The main advantages of the present application include: (1) Compared with the traditional CUT&RUN method, the CUT&RUNLT method of the present application significantly improves the experimental quality, and the number of peaks is significantly increased.

[0135] (2) The CUT&RUNLT method of the present application can release nucleic acid fragments that cannot be released by the traditional CUT&RUN method, thereby expanding the range of detection targets.

[0136] (3) The signal-to-noise ratio and sensitivity of the experimental result data of the CUT&RUNLT method of the present application are significantly better than those of the traditional method, and some experimental results are even better than those of the CUT&Tag technology, and the results of testing various nucleases are all better than those of the traditional CUT&RUN.

[0137] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out according to the conventional conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. The experimental materials and reagents involved in the present application can be obtained from commercial channels unless otherwise specified.

[0138] Experimental reagents The Concanavalin A magnetic beads (NovoNGS® Concanavalin A coated magnetic beads: N251), DNA extraction magnetic beads (Tagged DNA extraction magnetic beads: N245, NovoNGS® DNA clean-up magnetic beads: N240), and library construction kit (NovoNGS® DNA library preparation kit for Illumina® sequencing platform: N256; NovoNGS® Multiplex Indexing Primer Set 3 for Illumina® sequencing platform: E163) used in the present embodiment are all from Suzhou Coastal Protein Technology Co., Ltd. The primary antibodies used in the present embodiment are purchased from Merck Millipore (CTCF: 07-729), Active Motif (Pol-II: 39097; H3K4me3: 39159), and Cell Signaling Technology (CST) (rabbit IgG: 2729S). The cells used in the present embodiment are human 293F cells. The self-prepared reagent formulations of the buffers used in the experiment are shown in Table 1 (all taking 1 mL volume formulation as an example, the volume can be scaled up proportionally). Table 1

[0139] Experimental method: (1) Traditional CUT&RUN process The traditional CUT&RUN process in the prior art (reference document Skene, P., Henikoff, J. & Henikoff, S. Nat Protoc 13, 1006-1019 (2018), doi:10.1038 / nprot.2018.015) includes the following steps: 1. Preparation of cells 1-1) Collect fresh cells at room temperature and count, take the required cells in a new 1.5 mL centrifuge tube, centrifuge at 600 g for 5 min at room temperature, carefully discard the supernatant, and obtain the cell precipitate; 1-2) Add 1 mL of washing buffer to the cell precipitate of step 1-1), mix gently and centrifuge at 600 g for 5 min at room temperature, carefully discard the supernatant; 1-3) Add 90 μL of washing buffer to the cell precipitate in step 1-2) to resuspend the cells, and obtain the cell sample.

[0140] 2. Preparation and combination of ConA magnetic beads with cells 2-1) Take 10 μL of NovoNGS® Concanavalin A coated magnetic beads (hereinafter referred to as ConA magnetic beads) equilibrated to room temperature into a 1.5 mL centrifuge tube, place it on a magnetic stand for 2 min until the solution is clear, and discard the supernatant; 2-2) Add 100 μL binding buffer, mix well by pipetting; 2-3) Place the tube on a magnetic stand for 2 min until the solution is clear, discard the supernatant; 2-4) Repeat step 2-2), 2-3) once. 2-5) Take off the centrifuge tube, add 10 μL binding buffer, mix well by pipetting, and place on ice for later use; 2-6) Add 10 μL pretreated ConA magnetic beads to the 90 μL cell sample of step 1-3), mix gently by pipetting, and incubate for 10 min at room temperature on a rotary mixer to obtain a ConA magnetic bead-cell mixture.

[0141] 3. ConA magnetic bead-cell and primary antibody binding 3-1) Dilute the primary antibody with antibody buffer, 50 μL of primary antibody diluent is needed for each experimental sample, and the dilution ratio is specified in the instructions. Prepare the primary antibody diluent and place it on ice for later use; 3-2) Place the centrifuge tube containing the ConA magnetic bead-cell mixture from step 2-6) on a magnetic stand for 2 min, and after the magnetic beads and liquid are completely separated, carefully discard the supernatant. Take off the centrifuge tube, add 50 μL of primary antibody diluent, and incubate at room temperature for 2 h or at 4°C overnight.

[0142] 4. ConA magnetic bead-cell washing 4-1) Take off the centrifuge tube from step 3-2) after incubation of the primary antibody, place it on a magnetic stand for 2 min, and after the magnetic beads and liquid are completely separated, carefully discard the supernatant; 4-2) Take off the centrifuge tube from the magnetic stand, add 1 mL of Dig-washing solution, resuspend the ConA magnetic bead-cell mixture, mix gently by pipetting, and stand at room temperature for 2 min; 4-3) Place on a magnetic stand for 2 min, and after the magnetic beads and liquid are completely separated, carefully discard the supernatant; 4-4) Repeat steps 4-2), 4-3) twice.

[0143] 5. pAG-MNase binding and washing 5-1) Take off the centrifuge tube from the magnetic stand, add 50 μL of Dig-washing solution and pAG-MNase at a final concentration of 200 ng / mL, resuspend the ConA magnetic bead-cell mixture gently by pipetting, and incubate at 4°C for 1 h; 5-2) Place the centrifuge tube containing the incubated pAG-MNase on a magnetic stand for 2 min, and after the magnetic beads and liquid are completely separated, carefully discard the supernatant; 5-3) Remove the centrifuge tube from the magnetic stand, add 1 mL Dig-washing solution to resuspend the ConA magnetic beads-cell mixture, mix gently by blowing, and let stand at room temperature for 2 min; 5-4) Place on the magnetic stand for 2 min, and carefully discard the supernatant after the magnetic beads are completely separated from the liquid; 5-5) Repeat steps 5-3) and 5-4) twice.

[0144] 6. Fragmentation reaction 6-1) Remove the centrifuge tube from the magnetic stand, add 150 μL Dig-washing solution, mix the cells gently by blowing, and place on ice for standby; 6-2) Add 3 μL of 100 mM CaCl2 to the centrifuge tube of step 6-1), mix gently, immediately place on ice, and incubate for 30 min.

[0145] 7. Release of chromatin fragments 7-1) Add 100 μL of 2x termination buffer to the reaction completed centrifuge of step 6-2), mix by blowing or vortexing, and incubate at 37℃ in a metal or water bath for 10 min to release chromatin fragments into the supernatant.

[0146] 7-2) Centrifuge at 16000 g for 5 min at 4℃, place the centrifuge tube on the magnetic stand and let stand for 2 min, carefully transfer the supernatant to a new centrifuge tube after the magnetic beads are completely separated from the liquid, and the fragmented nucleic acid is obtained.

[0147] 8. DNA extraction 8-1) Add 2x volume of labeled DNA extraction magnetic beads (Novoprotein, Catalog No. N245) equilibrated to room temperature to the supernatant of step 7-2), mix gently by blowing, and let stand at room temperature for 5 min; 8-2) Transfer the PCR tube to the magnetic stand, let stand for 5 min to separate the magnetic beads from the liquid, and carefully discard the supernatant; 8-3) Add 200 μL of freshly prepared 80% ethanol to the PCR tube, let stand at room temperature for 1 min, then carefully discard the supernatant while the PCR tube is still on the magnetic stand; 8-4) Repeat step 8-3) once; 8-5) Place the PCR tube on the magnetic stand, open the PCR tube cap, and dry at room temperature (about 4 min) to allow the ethanol to evaporate completely; 8-6) Remove the PCR tube from the magnetic stand, add 22 μL of ddH2O to each tube, mix by blowing, and let stand at room temperature for 3 min; 8-7) Place the PCR tube on the magnetic stand for 2 min, and let the magnetic beads completely separate from the liquid. Carefully pipette 20 μL supernatant into a new PCR tube, which is the purified protein interaction nucleic acid fragment.

[0148] 9. Library construction The purified product is subjected to library construction using a DNA library preparation kit (NovoNGS® Illumina® sequencing platform, product number N256) and a multiplex index primer set 3 (NovoNGS® Illumina® sequencing platform, product number E163), and the operation is performed according to the instructions. 10. Library sequencing The purified library product is sent to a sequencing company for sequencing, and the sequencing results are analyzed on the Galaxy website (https: / / usegalaxy.org / ). The target protein binding map is viewed using IGV software.

[0149] (2) CUT&RUN LT process of the application: As shown in Figure 1 Compared with the traditional CUT&RUN process, the application changes the enzyme cutting and nucleic acid release conditions of the traditional CUT&RUN (low-temperature enzyme cutting chromatin DNA at 0-4℃ + high-temperature release of enzyme-cut fragments at 37℃), adds a protein nucleic acid fragment complex release agent to the enzyme cutting system, and adopts a low-temperature nucleic acid fragment release scheme, which can significantly improve the experimental quality. The signal-to-noise ratio and sensitivity of the experimental result data are much better than those of the traditional method. The CUT&RUN LT process of the application includes the following steps: 1. Preparation of cells 1-1) Collect fresh cells at room temperature and count them. Take the required cells into a new 1.5 mL centrifuge tube, centrifuge at 600 g for 5 min at room temperature, and carefully discard the supernatant to obtain the cell precipitate. 1-2) Add 1 mL of washing buffer to the cell precipitate of step 1-1), mix gently, and then centrifuge at 600 g for 5 min at room temperature. Carefully discard the supernatant. 1-3) Add 90 μL of washing buffer to the cell precipitate of step 1-2) to resuspend the cells, and obtain the cell sample.

[0150] 2. Preparation and combination of ConA magnetic beads with cells 2-1) Take 10 μL of ConA magnetic beads equilibrated to room temperature into a 1.5 mL centrifuge tube, place it on a magnetic stand for 2 min until the solution is clear, and discard the supernatant. 2-2) Add 100 μL of binding buffer and mix well. 2-3) Place the tube on the magnetic stand for 2 min until the solution is clear, discard the supernatant; 2-4) Repeat step 2-3) once; 2-5) Take off the centrifuge tube, add 10 μL binding buffer, mix well by pipetting, and place on ice for later use; 2-6) Add the 90 μL cell sample resuspended in step 1-3) to 10 μL of treated ConA magnetic beads, mix gently by pipetting, and place on a rotary mixer at room temperature for 10 min to obtain a ConA magnetic bead-cell mixture.

[0151] 3. ConA magnetic bead-cell and primary antibody binding 3-1) Dilute the primary antibody with antibody buffer, 50 μL of primary antibody diluent is needed for each experimental sample, and the dilution ratio is specified in the instructions. Prepare the primary antibody diluent and place it on ice for later use; 3-2) Place the centrifuge tube containing the ConA magnetic bead-cell mixture from step 2-6) on the magnetic stand for 2 min, carefully discard the supernatant after the magnetic beads and liquid are completely separated, take off the centrifuge tube, add 50 μL of primary antibody diluent, and incubate at room temperature for 2 h or at 4°C overnight.

[0152] 4. ConA magnetic bead-cell washing 4-1) Take off the centrifuge tube from step 3-2) after incubation with the primary antibody, place it on the magnetic stand for 2 min, and carefully discard the supernatant after the magnetic beads and liquid are completely separated; 4-2) Take off the centrifuge tube from the magnetic stand, add 200 μL of Dig-washing solution, resuspend the ConA magnetic bead-cell mixture, mix gently by pipetting, and stand at room temperature for 2 min; 4-3) Place on the magnetic stand for 2 min, carefully discard the supernatant after the magnetic beads and liquid are completely separated; 4-4) Repeat steps 4-2) and 4-3) once.

[0153] 5. pAG-MNase binding and washing 5-1) Take off the centrifuge tube from the magnetic stand, add 50 μL of Dig-washing solution and pAG-MNase at a final concentration of 200 ng / mL, resuspend the ConA magnetic bead-cell mixture gently by pipetting, and incubate at 4°C for 1 h; 5-2) Place the centrifuge tube containing the incubated pAG-MNase on the magnetic stand for 2 min, carefully discard the supernatant after the magnetic beads and liquid are completely separated; 5-3) Take off the centrifuge tube from the magnetic stand, add 200 μL of Dig-washing solution, resuspend the ConA magnetic bead-cell mixture, mix gently by pipetting, and stand at room temperature for 2 min; 5-4) Place the tube on the magnetic stand for 2 min, and carefully discard the supernatant after the magnetic beads are completely separated from the liquid; 5-5) Repeat steps 5-3) and 5-4) once.

[0154] 6. Fragmentation and chromatin release 6-1) Remove the centrifuge tube from the magnetic stand, add 50 μL of activation buffer, and gently mix the cells by blowing. Then, perform fragmentation and chromatin release in the following two schemes, which are collectively referred to as one-step method and two-step method, wherein, (1) Scheme one: one-step method includes the following steps: Place the tube in a 18℃ metal bath or PCR instrument, and incubate for 60 min to perform fragmentation and nucleic acid release at the same time.

[0155] (2) Scheme two: two-step method includes the following steps: Place the tube in a 4℃ metal bath or PCR instrument, and incubate for 40 min; add EDTA with a final concentration of 10 mM and EGTA with a final concentration of 2 mM to the reaction system, and incubate at 18℃ for 50 min; terminate the reaction and perform nucleic acid release.

[0156] 6-2) Place the reaction tube of step 6-1) on the magnetic stand for 2 min, and carefully transfer the supernatant to a new PCR tube after the magnetic beads are completely separated from the liquid, which is the fragmented nucleic acid.

[0157] 7. DNA extraction 7-1) Add 5 μL of 0.5 M EDTA, 4 μL of 10% SDS, and 1 μL of 20 mg / mL protease K to the supernatant obtained in step 6-2), mix well by blowing, and place at room temperature for 5 min; 7-2) Use magnetic bead purification to recover the nucleic acid fragments, add 2 times the volume of labeled DNA extraction magnetic beads equilibrated to room temperature to the system of the previous step, mix gently by blowing, and stand at room temperature for 5 min; 7-3) Transfer the PCR tube to the magnetic stand, stand for 5 min to separate the magnetic beads from the liquid, and carefully discard the supernatant; 7-4) Add 200 μL of freshly prepared 80% ethanol to the PCR tube, carefully discard the supernatant after standing at room temperature for 1 min, and still keep the PCR tube on the magnetic stand; 7-5) Repeat step 7-4) once; 7-6) Place the PCR tube on the magnetic stand, open the PCR tube cover, and dry at room temperature (about 4 min) to fully volatilize the ethanol; 7-7) Take the PCR tube off the magnetic stand, add 22 μL ddH2O to each tube, mix well by pipetting, and let it stand at room temperature for 3 min; 7-8) Place the PCR tube on the magnetic stand and let it stand for 2 min to completely separate the magnetic beads from the liquid. Carefully pipette 20 μL supernatant into a new PCR tube. The sample can be stored at -20°C or directly subjected to the next step of library construction.

[0158] 8. Library construction The purified product from the previous step was subjected to sequencing library construction using the DNA Library Preparation Kit (part number N256) and the NovoNGS® Illumina® Sequencing Platform Multiplex Index Primer Set 3 (part number E163) according to the instructions. 9. Library sequencing The purified sequencing library product was sent to a sequencing company for sequencing. The sequencing results were analyzed on the Galaxy website (https: / / usegalaxy.org / ). The target protein binding map was viewed using the IGV software.

[0159] Example 1: Effect of DMSO addition on CUT&RUN experimental results This example investigates the effect of DMSO addition on CUT&RUN experimental results. The experimental grouping and experimental flow design are shown in Table 2 below: Table 2

[0160] Experimental procedure: The following experiment was performed according to the CUT&RUN LT one-step method experimental procedure of the present application. For detailed operation, please refer to the CUT&RUN LT procedure description in the experimental method above. The following is a brief description of the steps: 1. Preparation of cells 1-1) Take 800,000 freshly harvested 293F cells and divide them into 8 aliquots in 1.5 mL centrifuge tubes, 100,000 cells per sample. Centrifuge at 600 g for 5 min at room temperature. Carefully discard the supernatant to obtain the cell pellet. 1-2) Add 1 mL of wash buffer to the cell pellet from step 1-1). Mix gently by pipetting, then centrifuge at 600 g for 5 min at room temperature. Carefully discard the supernatant to obtain the cell pellet. 1-3) Add 90 μL of wash buffer to the cell pellet in step 1-2) to resuspend the cells.

[0161] 2. Preparation and combination of ConA magnetic beads with cells 3. Combination of ConA magnetic bead-cells with primary antibody 4. Washing of ConA magnetic beads-cell 5. Binding and washing of pAG-MNase 6. Fragmentation reaction and chromatin release 6-1) The activation system in group 1, group 3, group 5 and group 7 in Table 2 above comprises: 50 μL of washing buffer, a final concentration of 10% DMSO and a final concentration of 1 mM CaCl2; The activation system in group 2, group 4, group 6 and group 8 comprises: 50 μL of washing buffer, a final concentration of 1 mM CaCl2, and no DMSO is added.

[0162] After gently mixing the cells by blowing, incubate at 18°C for 60 min on a PCR instrument to fragment and release nucleic acids at the same time.

[0163] 6-2) Place the reaction tube of step 6-1) on a magnetic stand and let it stand for 2 min, then carefully transfer the supernatant to a new PCR tube after the magnetic beads are completely separated from the liquid, which is the fragmented nucleic acid.

[0164] 7. DNA extraction 8. Library construction The same number of cycles is used for library amplification, and groups 1-8 above are all amplified for 14 cycles.

[0165] 9. Library sequencing The library product after magnetic bead purification is sent to a sequencing company for sequencing, and the sequencing results are analyzed on the Galaxy website (https: / / usegalaxy.org / ). The target protein binding map is viewed using IGV software, and the sequencing result analysis is shown in Figures 2-4 The effect of adding or not adding DMSO in different antibody groups on the number of library peaks is shown in Table 3.

[0166] Table 3

[0167] The results show that from the peak number in Table 3, after adding 10% DMSO, the peak number increases significantly. From the peak graph on IGV, the IgG group as a negative control is not affected by the addition of DMSO, while the three antibody groups Pol-II, CTCF and H3Kme3 groups without DMSO have partial peak missing phenomenon, that is, after adding DMSO, the original peak signal is enhanced, and the peak signal that cannot be detected is effectively detected. Figures 2-4the peaks in the antibody group Pol-II increased by more than 2.6 times compared with no addition of DMSO; the peaks in the antibody group CTCF increased by about 56% with the addition of DMSO; and the peaks in the antibody group H3Kme3 increased by about 29% with the addition of DMSO. Therefore, it can be inferred that the addition of DMSO at a final concentration of 10% in the activation step can promote the release of the target cleaved nucleic acid fragments into the extracellular supernatant.

[0168] Example 2: Effect of adding different concentrations of DMSO on test results This example investigates the effect of adding different concentrations of DMSO on test results. The specific experimental grouping design is shown in Table 4 below: Table 4

[0169] Experimental procedure: The following experiment was performed according to the CUT&RUN LT one-step method experimental procedure of the present application. For detailed operation, please refer to the CUT&RUN LT procedure description in the experimental method above. The following is a brief description of the steps: 1. Preparation of cells 1-1) Take 1.8 million freshly harvested 293F cells and divide them into 18 aliquots in 1.5 mL centrifuge tubes, 100,000 cells per sample, centrifuge at 600 g for 5 min at room temperature, and carefully discard the supernatant; 1-2) Add 1 mL of washing buffer to the cell pellet in step 1-1), mix gently by blowing, then centrifuge at 600 g for 5 min at room temperature, carefully discard the supernatant, and obtain the cell pellet; 1-3) Resuspend the cells in the cell pellet in step 1-2) with 90 μL of washing buffer.

[0170] 2. Preparation and combination of ConA magnetic beads with cells to obtain a ConA magnetic bead-cell mixture.

[0171] 3. Combination of ConA magnetic bead-cells with primary antibody.

[0172] 1) Dilute the Pol-II primary antibody with antibody buffer. Each experimental sample requires 50 μL of primary antibody diluent, which is placed on ice for standby; 2) Place the centrifuge tube containing the ConA magnetic bead-cell mixture combined in step 2 in a magnetic stand and stand for 2 min. After the magnetic beads and liquid are completely separated, carefully discard the supernatant, remove the centrifuge tube, add 50 μL of Pol-II primary antibody diluent, and rotate at room temperature for 2 h.

[0173] 4. Washing of ConA magnetic bead-cells 5. Binding and washing of pAG-MNase 6. Fragmentation and chromatin release 1) Activation systems for groups 1 and 10 in Table 4 above include: 50 μL wash buffer and a final concentration of 1 mM CaCl2, no DMSO added; Activation systems for groups 2 and 11 are 50 μL wash buffer, a final concentration of 0.3% DMSO and a final concentration of 1 mM CaCl2.

[0174] Activation systems for groups 3 and 12 are 50 μL wash buffer, a final concentration of 2.5% DMSO and a final concentration of 1 mM CaCl2.

[0175] Activation systems for groups 4 and 13 are 50 μL wash buffer, a final concentration of 5% DMSO and a final concentration of 1 mM CaCl2.

[0176] Activation systems for groups 5 and 14 are 50 μL wash buffer, a final concentration of 10% DMSO and a final concentration of 1 mM CaCl2.

[0177] Activation systems for groups 6 and 15 are 50 μL wash buffer, a final concentration of 15% DMSO and a final concentration of 1 mM CaCl2.

[0178] Activation systems for groups 7 and 16 are 50 μL wash buffer, a final concentration of 20% DMSO and a final concentration of 1 mM CaCl2.

[0179] Activation systems for groups 8 and 17 are 50 μL wash buffer, a final concentration of 25% DMSO and a final concentration of 1 mM CaCl2.

[0180] Activation systems for groups 9 and 18 are 50 μL wash buffer, a final concentration of 30% DMSO and a final concentration of 1 mM CaCl2.

[0181] 2) After gentle pipetting to mix the cells, groups 1-9 are incubated at 4°C for 60 min in a PCR machine, fragmenting and releasing nucleic acids at the same time; groups 10-18 are incubated at 20°C for 60 min in a PCR machine, fragmenting and releasing nucleic acids at the same time.

[0182] 3) Place the reaction tube from the previous step in a magnetic stand and let it stand for 2 min, then carefully transfer the supernatant to a new PCR tube, which is the fragmented nucleic acid.

[0183] 7. DNA extraction 8. Library construction The same number of cycles is used for library amplification, and groups 1-18 above are all amplified for 14 cycles.

[0184] 9. Library sequencing The library product after magnetic bead purification was sent to a sequencing company for sequencing, the sequencing results were analyzed on the Galaxy website (https: / / usegalaxy.org / ), the target protein binding map was viewed using IGV software, and the sequencing result analysis is shown in Table 5 and Figure 5 A- Figure 5 B.

[0185] Table 5

[0186] The results show that: in the data results of the same sequencing amount (1G) at two different temperatures (4℃ and 20℃), compared with the group without adding DMSO, after adding DMSO with a final concentration of 0.3%~30%, the peak number of the corresponding experimental group increased significantly (Table 5); in addition, in the IGV peak graph, compared with the group without adding DMSO, the peak signal after adding DMSO was enhanced, the background was weakened, and the signal-to-noise ratio was improved by more than one time (Fig. Figure 5 A- Figure 5 B). The results can show that within the range of DMSO final concentration of 0.3%~30%, the release of nucleic acid fragments can be enhanced.

[0187] Example 3: Effect of different temperatures in the activation step on the test This example investigates the effect of different temperatures in the activation step on the test, and the specific experimental grouping design is shown in Table 6: Table 6

[0188] Experimental steps: the following experiment refers to the CUT&RUN LT one-step method experimental process of the application, and the detailed operation is described in the above experimental method of the application CUT&RUN LT process. The following is a brief description of the steps: 1. Preparation of cells 1-1) Take 700,000 freshly harvested 293F cells, divide them into 7 aliquots in 1.5 mL centrifuge tubes, 100,000 cells / sample, centrifuge at 600 g for 5 min at room temperature, and carefully discard the supernatant; 1-2) Add 1 mL of washing buffer to the cell pellet in step 1-1), mix gently, then centrifuge at 600 g for 5 min at room temperature, carefully discard the supernatant, and obtain the cell pellet; 1-3) Resuspend the cells in 90 μL of washing buffer in step 1-2).

[0189] 2. Preparation and combination of ConA magnetic beads with cells 3. ConA magnetic beads-cell and primary antibody binding 1) Dilute Pol-II primary antibody with antibody buffer, 50 μL of primary antibody dilution is needed for each experimental sample, and keep it on ice for standby; 2) Place the centrifuge tube containing the ConA magnetic beads-cell mixture after the completion of step 2 on the magnetic stand for 2 min, and carefully discard the supernatant after the magnetic beads and liquid are completely separated. Take off the centrifuge tube, add 50 μL of Pol-II primary antibody dilution, and incubate at room temperature for 2 h.

[0190] 4. Washing of ConA magnetic beads-cell 5. Binding and washing of pAG-MNase 6. Fragmentation reaction and chromatin release 1) Group 1-7 in Table 6 above use an activation system including 50 μL of washing buffer, 10% DMSO with a final concentration, and 1 mM CaCl2 with a final concentration.

[0191] 2) After adding the activation system and gently mixing the cells, group 1 is incubated at 0°C (on ice), group 2 is incubated at 4°C in a PCR instrument, group 3 is incubated at 10°C in a PCR instrument, group 4 is incubated at 15°C in a PCR instrument, group 5 is incubated at 20°C in a PCR instrument, group 6 is incubated at 25°C in a PCR instrument, and group 7 is incubated at 30°C in a PCR instrument, while the nucleic acid is released simultaneously with the fragmentation for 60 min.

[0192] 3) Place the reaction centrifuge tube from the previous step on the magnetic stand for 2 min, and carefully transfer the supernatant to a new PCR tube after the magnetic beads and liquid are completely separated.

[0193] 7. DNA extraction 8. Library construction The library amplification uses the same number of cycles, and the above groups 1-7 are amplified for 14 cycles.

[0194] 9. Library sequencing The library product after magnetic bead purification is sent to a sequencing company for sequencing, and the sequencing results are analyzed on the Galaxy website (https: / / usegalaxy.org / ). The target protein binding map is viewed using IGV software, and the sequencing results are shown in Figure 6 , and the peak number results of different activation temperature groups in the 10% DMSO activation system are shown in Table 7: Table 7

[0195] The results show that in the experimental group with a final concentration of 10% DMSO (pol-II antibody), within the temperature range of 0°C to 30°C, the library yield increases with the increase of the activation temperature, and within the temperature range of 4°C to 20°C, the peak number shows an upward trend, while within the temperature range of 20°C to 30°C, the peak number shows a downward trend (as shown in Table 7). As shown in the IGV peak chart, Figure 6 ) at 0°C, the effect is slightly poor, the nucleic acid fragments cannot be fully released, the signal is weak, and the background is relatively high; within the temperature range of 4°C to 30°C, the peak type results with high signal-to-noise ratio can be obtained.

[0196] Example 4: Comparison of the process of CUT&RUN LT one-step activation and two-step activation with the traditional CUT&RUN method This Example 4 investigates the comparison of the results of the process of CUT&RUN LT one-step activation and two-step activation with the traditional CUT&RUN method, and the specific experimental grouping design is shown in Table 8 as follows: Table 8

[0197] Experimental steps: Different from the above examples, the experiments of this example are carried out according to the traditional CUT&RUN process, the CUT&RUN LT one-step method and the CUT&RUN LT two-step method in the CUT&RUN LT process of the present application respectively, and the detailed operations are described in the above experimental methods. The following is a brief description of the steps: Group 1 and Group 2 in Table 8 above are implemented by using the traditional CUT&RUN method, Group 3 and Group 4 are implemented by using the CUT&RUN LT one-step method, and Group 5 and Group 6 are implemented by using the CUT&RUN LT two-step method, and the specific operations are described as follows: 1. Preparation of cells 1-1) Take 600,000 freshly harvested 293F cells, divide them into 6 aliquots in 1.5 mL centrifuge tubes, 100,000 cells per sample, centrifuge at 600 g for 5 min at room temperature, carefully discard the supernatant, and obtain the cell pellet; 1-2) Add 1 mL of washing buffer to the cell pellet of step 1-1), mix gently, then centrifuge at 600 g for 5 min at room temperature, carefully discard the supernatant, and obtain the cell pellet; 1-3) Resuspend the cells in the cell pellet of step 1-2) by adding 90 μL of washing buffer.

[0198] The above 6 cell suspensions are used as Group 1 to Group 6, and Group 1 and Group 2 are implemented by using the traditional CUT&RUN method; 2. Preparation and combination of ConA magnetic beads and cells to obtain a mixture containing ConA magnetic beads and cells; 3. ConA magnetic beads-cell and primary antibody binding: 1) Group 1 in Table 8 above uses antibody buffer to dilute Pol-II primary antibody, 50 μL of primary antibody diluent is needed for each experimental sample, and is placed on ice for standby; Group 2 uses antibody buffer to dilute CTCF primary antibody, 50 μL of primary antibody diluent is needed for each experimental sample, and is placed on ice for standby; 2) Place the centrifuge tube containing the ConA magnetic bead-cell mixture combined in step 2 on the magnetic stand and stand for 2 min, then carefully discard the supernatant after the magnetic beads and liquid are completely separated, remove the centrifuge tube, and add 50 μL of corresponding Pol-II or CTCF primary antibody diluent to Group 1 and Group 2, respectively, and incubate at room temperature for 2 h; 4. ConA magnetic bead-cell washing; 5. pAG-MNase binding and washing; 6. Fragmentation reaction; 7. Chromatin fragment release; 8. DNA extraction; 9. Library construction: The library amplification uses the same number of cycles, and is amplified for 14 cycles; 10. Library sequencing.

[0199] For Group 3 and Group 4 in Table 8 above, the CUT&RUN LT one-step method is used, wherein steps 1-5 are basically the same as the above steps 1-5 of this embodiment, and the difference is the process after step 5, which includes the following steps: 6. Fragmentation reaction and chromatin release (one-step method): 1) The activation system used by Group 3 and Group 4 includes 50 μL of washing buffer, 10% DMSO with a final concentration, and 1 mM CaCl2 with a final concentration.

[0200] 2) After adding the activation system and gently blowing and mixing the cells, incubate at 18°C in a PCR instrument for 60 min, and perform nucleic acid release while fragmenting.

[0201] 3) Place the reaction centrifuge tube from the above step on the magnetic stand and stand for 2 min, then carefully transfer the supernatant to a new PCR tube to obtain the fragmented nucleic acid; 7. DNA extraction; 8. Library construction: The library amplification uses the same number of cycles, and is amplified for 14 cycles; 9. Library sequencing.

[0202] For group 5 and group 6 in Table 8 above, the CUT&RUNLT two-step method was implemented, wherein steps 1-5 involved were basically the same as the above-mentioned steps 1-5 of the embodiment, and the difference was the process after step 5, including the following steps: 6. Fragmentation reaction and chromatin release (two-step method) 1) Both group 5 and group 6 used an activation system of 50 μL washing buffer and a final concentration of 10% DMSO and a final concentration of 1 mM CaCl2; 2) After adding the activation system and gently blowing and mixing the cells, they were placed in a PCR instrument at 4°C and incubated for 40 min; 3) After incubation, a final concentration of 10 mM EDTA and 2 mM EGTA was added to the reaction system, and incubation was carried out at 18°C for 50 min to terminate the reaction and release the nucleic acid; 4) The reaction tube of the above step was placed in a magnetic stand and stood for 2 min, and after the magnetic beads were completely separated from the liquid, the supernatant was carefully transferred to a new PCR tube to obtain the fragmented nucleic acid.

[0203] 7. DNA extraction; 8. Library construction: The library amplification used the same number of cycles, and both were amplified for 14 cycles; 9. Library sequencing: The library product purified by magnetic beads was sent to a sequencing company for sequencing, and the sequencing results were analyzed on the Galaxy website (https: / / usegalaxy.org / ). The target protein binding map was viewed using IGV software, and the sequencing results are shown in Figures 7-8 , and the peak number determined by different CUT&RUN methods is shown in Table 9: Table 9

[0204] The results show that from the peak number in Table 9, the peak number in the two CUT&RUNLT schemes (CUT&RUNLT one-step method and CUT&RUNLT two-step method) is significantly higher than that of the traditional CUT&RUN experiment, and from the IGV peak graph, in the antibody Pol-II group and the antibody CTCF group, the signal-to-noise ratio of the CUT&RUNLT one-step method or the CUT&RUNLT two-step method of the present application is significantly improved, and the peak is more complete Figures 7-8 . It can be seen that under the conditions of low-temperature activation and the addition of a release-promoting agent (such as DMSO), both CUT&RUNLT implementation schemes (one-step method of simultaneous activation and release, and two-step method of separate activation and release) can obtain good results, which are significantly better than the results of the traditional CUT&RUN experiment.

[0205] Example 5: Effect of pAG-Benzonase Nuclease in Different Activation Systems This example investigates the effect of pAG-Benzonase nuclease in different activation systems with different DMF addition concentrations and compares the effects with pAG-MNase and pAG-DNase I. The specific experimental grouping design is shown in Table 10 below: Table 10

[0206] Experimental procedure: The following experiment refers to the CUT&RUN LT one-step method experimental procedure. For detailed operation, please refer to the description in the above experimental method section. The following is a brief description of the steps: 1. Preparation of cells: 1) Take 2.2 million freshly harvested 293F cells and divide them into 22 aliquots in 1.5 mL centrifuge tubes, 100,000 cells per sample, centrifuge at 600 g for 5 min at room temperature, and carefully discard the supernatant; 2) Add 1 mL of wash buffer to the cell pellet in the previous step, mix gently by blowing, then centrifuge at 600 g for 5 min at room temperature, and carefully discard the supernatant; 3) Add 90 μL of wash buffer to the cell pellet in the previous step to resuspend the cells; 2. Preparation and combination of ConA magnetic beads with cells; 3. Combination of ConA magnetic bead-cells with primary antibody: 1) Dilute Pol-II primary antibody with antibody buffer for groups 1-11 in Table 10, 50 μL of primary antibody diluent is required for each experimental sample, and store on ice; dilute CTCF primary antibody with antibody buffer for groups 12-22, 50 μL of primary antibody diluent is required for each experimental sample, and store on ice; 2) Place the centrifuge tube containing the ConA magnetic bead-cell mixture from step 2 in the magnetic stand and stand for 2 min, then carefully discard the supernatant after the magnetic beads and liquid are completely separated, remove the centrifuge tube, and add 50 μL of corresponding Pol-II or CTCF primary antibody diluent according to groups 1-11 and 12-22, respectively, and incubate at room temperature for 2 h; 4. Washing of ConA magnetic bead-cells; 5. Combination and washing of pAG-Benzonase, pAG-MNase, and pAG-DNase I: Groups 1-9 and 12-20 in Table 10 use pAG-Benzonase nuclease, groups 10 and 21 use pAG-MNase nuclease, and groups 11 and 22 use pAG-DNase I nuclease: 5-1) Remove the centrifuge tubes from the magnetic stand, add 50 μL Dig- wash solution and final concentration 200 ng / mL of pAG-Benzonase for group 1~9, group 12~20; add 50 μL Dig-wash solution and final concentration 200 ng / mL of pAG-MNase for group 10 and 21; add 50 μL Dig-wash solution and final concentration 200 ng / mL of pAG-DNase I for group 11 and 22, gently resuspend the ConA magnetic beads-cell mixture, and incubate at 4°C for 1 h; 5-2) Place the centrifuge tubes incubated with pAG-Benzonase, pAG-MNase, and pAG-DNase I, respectively, on the magnetic stand and stand for 2 min. After the magnetic beads and liquid are completely separated, carefully discard the supernatant; 5-3) Remove the centrifuge tubes from the magnetic stand, add 200 μL Dig- wash solution, resuspend the ConA magnetic beads-cell mixture, gently mix, and stand at room temperature for 2 min; 5-4) Place on the magnetic stand and stand for 2 min. After the magnetic beads and liquid are completely separated, carefully discard the supernatant; 5-5) Repeat steps 5-3) and 5-4) once.

[0207] 6. Fragmentation reaction and chromatin release 1) Group 1 and 12 activated system is 50 μL washing buffer, final concentration of 1 mM MgCl2, no DMF added; Group 2 and 13 activated system is 50 μL washing buffer, final concentration of 0.3% DMF and final concentration of 1 mM MgCl2; Group 3 and 14 activated system is 50 μL washing buffer, final concentration of 2.5% DMF and final concentration of 1 mM MgCl2; Group 4 and 15 activated system is 50 μL washing buffer, final concentration of 5% DMF and final concentration of 1 mM MgCl2; Group 5 and 16 activated system is 50 μL washing buffer, final concentration of 10% DMF and final concentration of 1 mM MgCl2; Group 6 and 17 activated system is 50 μL washing buffer, final concentration of 15% DMF and final concentration of 1 mM MgCl2; Group 7 and 18 activated system is 50 μL washing buffer, final concentration of 20% DMF and final concentration of 1 mM MgCl2; Group 8 and 19 activated system is 50 μL washing buffer, final concentration of 25% DMF and final concentration of 1 mM MgCl2; Group 9 and 20 activated system is 50 μL washing buffer, final concentration of 30% DMF and final concentration of 1 mM MgCl2; Group 10 and 21, activated system is 50 μL washing buffer, final concentration of 15% DMF and final concentration of 1 mM CaCl2; Group 11 and 22, activated system is 50 μL washing buffer, final concentration of 15% DMF and final concentration of 1 mM MgCl2.

[0208] 2) After gentle blowing and mixing the cells, all groups were incubated at 18°C for 60 min on a PCR instrument for fragmenting and releasing nucleic acid at the same time; 3) The reaction tube of the previous step was placed on a magnetic stand for 2 min, and after the magnetic beads were completely separated from the liquid, the supernatant was carefully transferred to a new PCR tube, which was the fragmented nucleic acid; 7. DNA extraction; 8. Library construction: The library amplification used the same number of cycles, and all were amplified for 14 cycles; 9. Library sequencing: The library product after magnetic bead purification was sent to a sequencing company for sequencing. The sequencing results were analyzed on the Galaxy website (https: / / usegalaxy.org / ). The target protein binding map was viewed using IGV software. The sequencing results are shown in Tables 11 and Figure 9 A- Figure 9 B.

[0209] Table 11

[0210] The results show that: in the experimental results of Pol-II and CTCF antibodies, the peak number of the two antibody groups after adding DMF with a final concentration of 0.3% to 30% is significantly increased in the same sequencing amount (1G) of data results compared with the group without adding cell membrane permeabilizer DMF (Table 11); in addition, it can be observed in the IGV peak graph that the peak signal of the two antibody groups is enhanced after adding DMF, the background is weakened, and the signal-to-noise ratio is improved (Figs. 11A and 11B). Figure 9 A- Figure 9 B). The results can show that in the range of 0.3% to 30% of the final concentration of DMF, the release of nucleic acid fragments can be enhanced.

[0211] In the activation system with the addition of 15% DMF, the peak numbers of the three nucleases pAG-Benzonase and pAG-MNase, and pAG-DNase I are similar, and the IGV peak graph shows that the peak type and signal-to-noise ratio of the results of the three nucleases are similar as a whole.

[0212] Example 6: Effect of the addition of DMSO on the results of CUT&RUN experiment of cell nucleus sample This example investigates the effect of the addition of DMSO on the results of CUT&RUN experiment of cell nucleus sample, and the specific experimental grouping design is shown in Table 12: Table 12

[0213] Experimental steps: the following experiment refers to the CUT&RUN LT one-step method experimental process, and the detailed operation is described in the description of the above experimental method part. The following is a brief description of the steps: Preparation of cell nucleus Take 800,000 fresh harvested 293F cells, divide them into 8 parts in 1.5 mL centrifuge tubes, 100,000 cells per sample, centrifuge at 600 g for 5 min at room temperature, and carefully discard the supernatant; Add 1 mL lysis buffer (formula see experimental method part) to the cell pellet, mix gently by blowing, and incubate on ice for 5 min to lyse the cells; Centrifuge at 600 g for 5 min at 4°C, carefully discard the supernatant, and obtain the cell nucleus pellet; Add 1 mL washing buffer to the cell nucleus pellet of the previous step, mix gently by blowing, and centrifuge at 600 g for 5 min at room temperature, carefully discard the supernatant, and obtain the cell nucleus pellet; Add 90 μL washing buffer to the cell nucleus pellet in the previous step to resuspend the cell nucleus.

[0214] Preparation of ConA magnetic beads and combination with cell nucleus ConA magnetic bead-cell nucleus and primary antibody combination ConA magnetic beads - washing of cell nuclei binding and washing of pAG-MNase Fragmentation reaction and chromatin release The activation systems for groups 1 / 3 / 5 / 7 in Table 12 above consisted of 50 μL of wash buffer, 10% DMSO, and 1 mM CaCl2. The activation systems for groups 2 / 4 / 6 / 8 consisted of 50 μL of wash buffer, 1 mM CaCl2, and no DMSO was added. After gently mixing the cells by pipetting, the cells were incubated at 18°C ​​for 60 min in a PCR instrument, releasing nucleic acids simultaneously with cell fragmentation.

[0215] Place the centrifuge tube from the previous step on a magnetic rack and let it stand for 2 minutes. After the magnetic beads have completely separated from the liquid, carefully transfer the supernatant to a new PCR tube to obtain the fragmented nucleic acid.

[0216] DNA extraction Library Construction Library amplification used the same number of cycles, with groups 1-8 all undergoing 14 cycles of amplification.

[0217] Library sequencing The purified library products were sent to a sequencing company for sequencing. The sequencing results were analyzed on the Galaxy website (https: / / usegalaxy.org / ). Target protein binding maps were viewed using IGV software. The sequencing results are shown below. Figure 10 A- Figure 10 As shown in C, the number of library peaks with and without DMSO addition in the CUT&RUN experiment of cell nuclear samples is shown in Table 13: Table 13

[0218] The results showed that, based on the peak count in Table 13, the number of nuclear-derived peaks increased significantly after adding 10% DMSO compared to the group without DMSO. The peak plot on IGV showed that, as a negative control, the IgG group was unaffected by the addition of DMSO, while the three antibody groups (Pol-II, CTCF, and H3Kme3) showed partial peak loss in the DMSO-free groups. Figure 10 A- Figure 10 (as indicated by the arrow in C), therefore, adding DMSO to a final concentration of 10% during the activation step can promote the release of targeted cleaved nucleic acid fragments into the extranuclear supernatant.

[0219] Example 7: Effect of different protein-nucleic acid fragment release agents on CUT&RUN experimental results This example investigates the effect of the addition of different protein nucleic acid fragment complex release agents, Tween-20, NP40 (or IGEPAL CA-630), Triton X-100, CHAPS, sodium deoxycholate, on the results of CUT&RUN experiments. The specific experimental grouping design is shown in Table 14: Table 14

[0220] Experimental procedure: The following experiment refers to the CUT&RUN LT one-step method experimental procedure. For detailed operation, please refer to the description above. The following is a brief description of the steps: 1. Preparation of cells 1) Take 1 million freshly harvested 293F cells, aliquot into 12 1.5 mL centrifuge tubes, 100,000 cells per sample, centrifuge at 600 g for 5 min at room temperature, carefully discard the supernatant; 2) Add 1 mL of wash buffer to the cell pellet in the previous step, mix gently by blowing, then centrifuge at 600 g for 5 min at room temperature, carefully discard the supernatant, and obtain the cell pellet; 3) Add 90 μL of wash buffer to the cell pellet in the previous step to resuspend the cells; 2. Preparation and combination of ConA magnetic beads with cells; 3. Combination of ConA magnetic bead-cells with primary antibody: 1) Dilute the Pol-II primary antibody using antibody buffer. Each experimental sample requires 50 μL of primary antibody diluent, which is placed on ice for standby; 2) Place the centrifuge tube containing the ConA magnetic bead-cell mixture from step 2 on the magnetic stand and stand for 2 min. After the magnetic beads and liquid are completely separated, carefully discard the supernatant, remove the centrifuge tube, add 50 μL of Pol-II primary antibody diluent, and rotate incubate at room temperature for 2 h; 4. Washing of ConA magnetic bead-cells; 5. Combination and washing of pAG-MNase; 6. Fragmentation reaction and chromatin release: 1) Group 1~6 in Table 14 above respectively 50 μL of wash buffer, final concentration of 0.05% of digitalis saponin or Tween-20 or NP40 or TritonX-100 or CHAPS or sodium deoxycholate, and final concentration of 1 mM of CaCl2, and group 7~12 50 μL of wash buffer, final concentration of 0.5% of digitalis saponin or Tween-20 or NP40 or TritonX-100 or CHAPS or sodium deoxycholate, and final concentration of 1 mM of CaCl2. After gently blowing and mixing the cells, incubate at 18℃ on the PCR instrument for 60 min, and release the nucleic acid while fragmenting; 2) Place the centrifuge tube of the above reaction on the magnetic stand and stand for 2 min, then carefully transfer the supernatant to a new PCR tube after the magnetic beads are completely separated from the liquid, which is the fragmented nucleic acid; 7. DNA extraction; 8. Library construction: The same number of cycles is used for library amplification, and groups 1~12 are amplified for 14 cycles; 9. Library sequencing: The library product after magnetic bead purification is sent to a sequencing company for sequencing, and the sequencing results are analyzed on the Galaxy website (https: / / usegalaxy.org / ). The target protein binding map is viewed using IGV software, and the sequencing result analysis is as shown in Figure 11 , and the number of library peaks after adding different detergents is shown in Table 15: Table 15

[0221] The results show that from the peak number in Table 15, the peak number of the group adding a concentration of 0.05% of the protein nucleic acid fragment complex release agent is slightly lower than that of the group adding a concentration of 0.5%. From the IGV peak graph, after adding different protein nucleic acid fragment complex release agents, CUT&RUN results with high signal-to-noise ratio can be obtained Figure 11 , so the above common protein nucleic acid fragment complex release agents can achieve the effect of promoting the release of protein nucleic acid fragment complex.

[0222] Example 8: Effect of different release promoters on CUT&RUN experimental results This example investigates the effect of adding different release promoters streptolysin O and melittin on the results of CUT&RUN experiments. The specific experimental grouping design is shown in Table 16: Table 16

[0223] The experimental process of this example is basically the same as that of Example 7, except for step 6, which is as follows: 6. Fragmentation reaction and chromatin release 1) Group 1 activation system in Table 16 above is 50 μL wash buffer, final concentration of 5% DMSO, and final concentration of 1 mM CaCl2; Group 2 activation system is 50 μL wash buffer, final concentration of 10 ng / mL streptolysin O (hereinafter referred to as SLO), and final concentration of 1 mM CaCl2; Group 3 activation system is 50 μL wash buffer, final concentration of 50 ng / mL SLO, and final concentration of 1 mM CaCl2; Group 4 activation system is 50 μL wash buffer, final concentration of 100 ng / mL SLO, and final concentration of 1 mM CaCl2; Group 5 activation system is 50 μL wash buffer, final concentration of 0.01 μM melittin, and final concentration of 1 mM CaCl2; Group 6 activation system is 50 μL wash buffer, final concentration of 0.1 μM melittin, and final concentration of 1 mM CaCl2; Group 7 activation system is 50 μL wash buffer, final concentration of 1 μM melittin, and final concentration of 1 mM CaCl2. After gently blowing and mixing the cells, incubate at 18°C on a PCR instrument for 60 min to release the nucleic acid while fragmenting.

[0224] 2) Place the centrifuge tube of the previous reaction on a magnetic stand and stand for 2 min, then carefully transfer the supernatant to a new PCR tube after the magnetic beads and liquid are completely separated, which is the fragmented nucleic acid.

[0225] The subsequent experimental process is the same as that in Example 7. The sequencing result analysis is shown in Figure 12 , and the number of library peaks after adding different release promoters is shown in Table 17: Table 17

[0226] The results show that from the peak number in Table 17, the peak number of Group 1 with single addition of 5% DMSO has no obvious difference from that of Groups with addition of streptolysin O or melittin, and at three different concentrations, the peak signal after addition of streptolysin O or melittin can be observed in the IGV peak graph, and the signal-to-noise ratio is similar Figure 12 , so the addition of different concentrations of release promoters: streptolysin O and melittin can promote the release of target cleavage nucleic acid fragments to the extracellular supernatant.

[0227] Example 9: Effect of different release promoter combinations on CUT&RUN experimental results This example investigates the effect of the addition of different release promoter combinations on the CUT&RUN experimental results, and the specific experimental grouping design is shown in Table 18: Table 18

[0228] The experimental process of this example is basically the same as that of Example 8, except that step 6 is different, and the specific process is shown as follows: 6. Fragmentation reaction and chromatin release 1) The activation system of group 1 in Table 18 above is 50 μL of washing buffer, a final concentration of 5% DMSO, and a final concentration of 1 mM CaCl2; the activation system of group 2 is 50 μL of washing buffer, a final concentration of 5% DMSO and a final concentration of 0.05% digitonin, and a final concentration of 1 mM CaCl2; the activation system of group 3 is 50 μL of washing buffer, a final concentration of 5% DMSO and a final concentration of 0.05% Tween-20, and a final concentration of 1 mM CaCl2; the activation system of group 4 is 50 μL of washing buffer, a final concentration of 5% DMSO and a final concentration of 0.05% NP40, and a final concentration of 1 mM CaCl2; the activation system of group 5 is 50 μL of washing buffer, a final concentration of 5% DMSO and a final concentration of 0.05% Triton X-100, and a final concentration of 1 mM CaCl2; the activation system of group 6 is 50 μL of washing buffer, a final concentration of 5% DMSO and a final concentration of 0.05% CHAPS, and a final concentration of 1 mM CaCl2; the activation system of group 7 is 50 μL of washing buffer, a final concentration of 5% DMSO and a final concentration of 0.05% sodium deoxycholate, and a final concentration of 1 mM CaCl2. The activation system of group 8 is 50 μL of washing buffer, a final concentration of 0.05% NP40 and a final concentration of 0.05% digitonin, and a final concentration of 1 mM CaCl2; the activation system of group 9 is 50 μL of washing buffer, a final concentration of 0.05% NP40 and a final concentration of 0.05% Tween-20; the activation system of group 10 is 50 μL of washing buffer, a final concentration of 0.05% NP40 and a final concentration of 0.05% Triton X-100; the activation system of group 11 is 50 μL of washing buffer, a final concentration of 0.05% NP40 and a final concentration of 0.05% CHAPS; the activation system of group 12 is 50 μL of washing buffer, a final concentration of 0.05% NP40 and a final concentration of 0.05% sodium deoxycholate; After gently blowing and mixing the cells, incubate at 18°C on a PCR instrument for 60 min to fragment and release the nucleic acids at the same time.

[0229] 2) Place the reaction centrifuge tube from the previous step on a magnetic stand and let it stand for 2 min until the magnetic beads and the liquid are completely separated. Carefully transfer the supernatant to a new PCR tube to obtain the fragmented nucleic acids.

[0230] The subsequent experimental process is the same as that of Example 8. The sequencing result analysis is as follows: Figure 13As shown, the number of peaks after adding different combinations of release-promoting agents is shown in Table 19: Table 19

[0231] The results show that, in terms of the number of peaks in Table 19, the number of peaks in the group with the addition of 5% DMSO alone is not significantly different from the number of peaks in each group with the addition of two combinations of release-promoting agents, and the peak signals can be observed in the IGV peak graph, and the signal-to-noise ratio is similar (Figure 19) Figure 13 Therefore, the addition of different combinations of release-promoting agents in the activation step can promote the release of targeted cleaved nucleic acid fragments into the extracellular supernatant.

[0232] Example 10: Effect of different temperature gradients and addition of release-promoting agents after activation on the results of CUT&RUN experiments This example investigates the effect of different temperature gradients and the addition of release-promoting agents after activation on the results of CUT&RUN experiments. The specific experimental grouping design is shown in Table 20: Table 20

[0233] Experimental steps: The following experiments refer to the CUT&RUN LT one-step method experimental procedure. For detailed operations, please refer to the description in the above experimental method section. The following is a brief description of the steps: 1. Preparation of cells: 1) Take 600,000 freshly harvested 293F cells and divide them into 6 aliquots in 1.5 mL centrifuge tubes, 100,000 cells per sample, centrifuge at 600 g for 5 min at room temperature, and carefully discard the supernatant; 2) Add 1 mL of wash buffer to the cell pellet from the previous step, mix gently by blowing, then centrifuge at 600 g for 5 min at room temperature, and carefully discard the supernatant; 3) Resuspend the cells in 90 μL of wash buffer in the cell pellet from the previous step; 2. Preparation and combination of ConA magnetic beads with cells; 3. Combination of ConA magnetic bead-cells with primary antibodies: 1) For group 1, group 3, and group 5 in Table 20, dilute the Pol-II primary antibody with antibody buffer, 50 μL of primary antibody diluent is required for each experimental sample, and place it on ice for standby; For group 2, group 4, and group 6, dilute the CTCF primary antibody with antibody buffer, 50 μL of primary antibody diluent is required for each experimental sample, and place it on ice for standby; 2) Place the centrifuge tube containing the ConA magnetic beads-cell mixture from step 2 on the magnetic stand and let it stand for 2 min. After the magnetic beads and liquid are completely separated, carefully discard the supernatant, remove the centrifuge tube, and add 50 μL of the corresponding Pol-II or CTCF primary antibody diluent according to the group number. Incubate at room temperature for 2 h with rotation; 4. Washing of ConA magnetic beads-cells; 5. Binding and washing of pAG-MNase; 6. Fragmentation reaction and chromatin release; 1) The activation system of groups 1-4 is 50 μL of washing buffer, a final concentration of 1 mM CaCl2, and a final concentration of 5% DMSO. The activation system of groups 5 and 6 is 50 μL of washing buffer and a final concentration of 1 mM CaCl2, without release promoter.

[0234] 2) After gently blowing and mixing the cells, all groups are subjected to fragmentation and release on a PCR instrument according to the following program: Group 1 and group 2: according to the following program: 0°C, incubate for 20 min, 4°C, incubate for 20 min, 10°C, incubate for 20 min; Group 3 and group 4: activation temperature 18°C, incubate for 60 min, nucleic acid release is performed at the same time as fragmentation; Group 5 and group 6: activation temperature 18°C, incubate for 40 min, add a final concentration of 5% DMSO to the system, 18°C, incubate for 20 min, and release the nucleic acid fragments; 3) Place the reaction centrifuge tube from the previous step on the magnetic stand and let it stand for 2 min. After the magnetic beads and liquid are completely separated, carefully transfer the supernatant to a new PCR tube, which is the fragmented nucleic acid; 7. DNA extraction; 8. Library construction: The same number of cycles is used for library amplification, and all are amplified for 14 cycles; 9. Library sequencing: The library product after magnetic bead purification is sent to a sequencing company for sequencing. The sequencing results are analyzed on the Galaxy website (https: / / usegalaxy.org / ). The target protein binding map is viewed using IGV software. The sequencing result analysis is shown in Figure 14 The peak number of different library groups is shown in Table 21: Table 21

[0235] The results showed that, based on the peak counts in Table 21, the number of peaks in the three-stage, temperature-gradient reaction using the CUT&RUNLT one-step method was not significantly different from that of the standard CUT&RUNLT one-step method (with the activation system premixed with the release promoter DMSO) and the CUT&RUNLT one-step method (first activating with metal ions for nucleic acid fragmentation, then adding the release promoter to aid the diffusion of the protein-nucleic acid fragment complex). Both antibodies, Pol-II and CTCF, showed similar peak signals and signal-to-noise ratios in the IGV peak diagram. Figure 14 (Top three lines: Pol-II antibody results; bottom three lines: CTCF antibody results) This indicates that there is no significant difference among the three methods; all three can obtain target protein action site maps with high signal-to-noise ratios. It can also be concluded that the release promoter, whether added during the activation step or added alone after activation, can promote the release of targeted cleaved nucleic acid fragments into the extracellular supernatant.

[0236] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method of constructing a library of nucleic acids, characterized by, The method comprises the following steps: S1. providing a cell or nucleus sample; S2. washing or resuspending the cell or nucleus sample to obtain a cell or nucleus suspension; S3. pretreating the cell or nucleus suspension to obtain a cell or nucleus precipitate; S4. treating the cell or nucleus precipitate with an antibody to obtain an antibody-cell or nucleus complex; S5. treating the antibody-cell or nucleus complex with a nuclease to obtain a nuclease-antibody-cell or nucleus complex; S6. performing fragmentation treatment on the nuclease-antibody-cell or nucleus complex with a protein nucleic acid fragment complex releasing agent in an activation release reaction system containing a metal ion buffer at a temperature T to release nucleic acid fragments; wherein the temperature T is 0-30°C; and the treatment time t is 20-240 min; S7. extracting the released nucleic acid fragments to prepare a nucleic acid library.

2. The method of claim 1, wherein, The protein nucleic acid fragment complex releasing agent is selected from the group consisting of a non-ionic surfactant, an ionic surfactant, a bioactive perforator, a polar solvent, or a combination thereof.

3. The method of claim 1, wherein, The protein nucleic acid fragment complex releasing agent is selected from the group consisting of DMSO, SMF, Tween-20, NP40, Triton X-100, CHAPS, sodium deoxycholate, streptolysin O, melittin, or a combination thereof.

4. The method of claim 1, wherein, The step S6 comprises any one of the following steps: S61. performing fragmentation treatment on the nuclease-antibody-cell or nucleus complex with a protein nucleic acid fragment complex releasing agent in an activation release reaction system at a temperature T1 for a time t1 to release nucleic acid fragments; S62. performing fragmentation treatment on the nuclease-antibody-cell or nucleus complex with a protein nucleic acid fragment complex releasing agent in an activation release reaction system at a temperature T2 for a time t2 to release nucleic acid fragments; adding a metal chelator, treating at a temperature T3 for a time t3 to terminate the reaction and further release nucleic acid fragments; S63. treating the nuclease-antibody-cell or nucleus complex in a metal ion buffer without a protein nucleic acid fragment complex releasing agent at a temperature T4 for a time t4, and then performing the reaction procedure of the step S61; or S64. treating the nuclease-antibody-cell or nucleus complex in a metal ion buffer without a protein nucleic acid fragment complex releasing agent at a temperature T4 for a time t4, and then performing the reaction procedure of the step S62.

5. The method of claim 1, wherein, The temperature T of the nucleic acid fragmentation treatment in the step S6 is a fixed temperature value or a combination of multiple temperature values; when the temperature is a combination of multiple temperature values, the process comprises performing the reaction at different temperatures respectively.

6. The method of claim 1, wherein, In the step S6, the activation release reaction system comprises a protein nucleic acid fragment complex releasing agent and a metal ion buffer with a concentration of 0.01 mM-10 mM.

7. The method of claim 6, wherein, The metal ions in the metal ion buffer are selected from the group consisting of calcium ions, magnesium ions, manganese ions, iron ions, zinc ions, cobalt ions, or a combination thereof.

8. The method of claim 1, wherein, The nuclease has endonuclease activity, can cleave single-stranded or double-stranded nucleic acids without sequence dependence, and has metal ion activation characteristics.

9. A kit for constructing a nucleic acid library using the method of claim 1, comprising: (1) a protein nucleic acid fragment complex release agent; (2) a metal ion buffer; (3) a cell washing buffer; and (4) a fragmentation reaction reagent.

10. The kit of claim 9, wherein The protein nucleic acid fragment complex release agent is selected from the group consisting of non-ionic surfactants, ionic surfactants, biologically active perforators, polar solvents, or a combination thereof; The metal ions in the metal ion buffer are selected from the group consisting of calcium ions, magnesium ions, manganese ions, iron ions, zinc ions, cobalt ions, or a combination thereof; The fragmentation reaction reagent is selected from the group consisting of nucleases, magnetic beads, antibodies, or a combination thereof.

11. A method of measuring protein-nucleic acid interactions, characterized in that, comprising: (S1) constructing a nucleic acid library using the method of claim 1; and (S2) sequencing the nucleic acid library to generate a plurality of sequencing reads for measuring protein-nucleic acid interactions.

Images