A method for studying nucleic acid-protein interactions

Abstract

This invention provides a method for studying nucleic acid-protein interactions, belonging to the field of biotechnology. Specifically, this invention improves the reaction system formulation by increasing the permeability of cell membranes or nuclear membranes and lowering the release temperature of nucleic acid fragments, thereby improving the signal-to-noise ratio, sensitivity, and resolution of experimental data results, and enhancing the detection effect of protein-nucleic acid interaction studies.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for studying nucleic acid-protein interactions. Background Technology

[0002] Epigenetics and epitranscription are key regulatory mechanisms shaping gene expression and protein translation. Epigenetic regulation refers to the process of regulating the content and function of nucleic acids or proteins within cells through epigenetic modifications (such as DNA methylation, modification of chromatin histones, and regulation of non-coding RNA) without altering the DNA sequence. All epigenetic regulation shares a common goal: adjusting chromatin state to regulate gene expression. In eukaryotic genomes, nucleosomes are the basic structural units of chromatin, composed of DNA encapsulating four core histones. Regions with fewer nucleosomes facilitate the binding and interaction of regulatory elements such as transcription factors with promoters and enhancers in these regions, thereby regulating gene expression. Dense nucleosomes contain relatively closed structural regions related to gene expression. Disruption of epigenetically controlled gene expression can lead to various diseases; therefore, studying protein-nucleic acid interaction patterns is crucial for understanding subsequent disease development.

[0003] Affinity-targeted methods for studying protein-DNA interactions primarily involve specifically capturing target proteins or DNA to study their interactions. These methods typically rely on antibodies, tags, or other affinity reagents to enrich the target molecules, such as ChIP, CUT&Tag, CUT&RUN, etc.

[0004] CUT&RUN technology was first developed and published by Steven Henikoff's laboratory in 2017. Inspired by early chromatin cleavage techniques (such as ChIC), it incorporates the specific cleavage capability of micrococcal nuclease (MNase). Its core principle is to perform targeted cleavage directly within the cell nucleus, avoiding the background noise caused by chromatin cross-linking and fragmentation in ChIP. MNase cleaves DNA near the target protein binding site, releasing the protein-DNA complex into the cell / nucleus. Nucleic acid extraction from the released products and construction of nucleic acid sequencing libraries can yield nucleic acid-protein interaction maps; proteomic analysis can also be performed to obtain proteomics data. While CUT&RUN technology offers a higher signal-to-noise ratio compared to ChIP-seq, it suffers from lower signal-to-noise ratios, lower sensitivity, and poorer experimental stability compared to CUT&Tag. These factors limit the application scope of CUT&RUN technology, making its application range far smaller than that of CUT&Tag.

[0005] Therefore, there is an urgent need in this field to develop methods that can improve and enhance the performance of CUT&RUN technology. Summary of the Invention

[0006] The purpose of this invention is to provide a novel experimental method for targeted cleavage of nucleic acids by nucleases. This invention employs a scheme to increase the permeability of the cell membrane (or nuclear membrane) and reduce the release temperature of nucleic acid fragments, thereby solving 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 improving the application effect of CUT&RUN and similar technologies.

[0007] In a first aspect of the present invention, a method for constructing a nucleic acid library is provided, comprising:

[0008] (S1) Provide a sample of a cell or cell nucleus;

[0009] (S2) Wash or resuspend the cell or nucleus sample to obtain a cell or nucleus suspension;

[0010] (S3) The cell or cell nucleus suspension is pretreated to obtain cell or cell nucleus precipitate;

[0011] (S4) Treat the cell or cell nucleus precipitate with the antibody to obtain an antibody-cell or cell nucleus complex;

[0012] (S5) Treat the antibody-cell or cell nucleus complex with a nuclease to obtain a nuclease-antibody-cell or cell nucleus complex;

[0013] (S6) Under temperature T (°C), in an activation and release reaction system containing metal ion buffer, the nuclease-antibody-cell or cell nucleus complex is treated with a protein-nucleic acid fragment release agent (hereinafter referred to as the release agent) for time t (min) to perform nucleic acid fragmentation and release of nucleic acid fragments, wherein the temperature T is 0~30°C, preferably 4~20°C, more preferably 18°C; the treatment time t is 20~240 min, preferably 40~90 min, more preferably 60 min;

[0014] (S7) Extract the released nucleic acid fragments and prepare a nucleic acid library.

[0015] In another preferred embodiment, the pretreatment method includes using magnetic beads, centrifugation, or other methods of grasping cells or cell nuclei.

[0016] In another preferred embodiment, the other method of grasping cells or cell nuclei includes: grasping cells or cell nuclei on some solid surface by means of coupling proteins.

[0017] In another preferred embodiment, the protein-nucleic acid fragment release agent is selected from the group consisting of: nonionic surfactants, ionic surfactants, bioactive pore-forming agents, polar solvents, or combinations thereof.

[0018] In another preferred embodiment, the nonionic surfactant is selected from the group consisting of polysorbate compounds, alkylphenol polyoxyethylene ether compounds, NP40 or IGEPA CA-630, digitalis saponins, or combinations thereof.

[0019] 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 combinations thereof.

[0020] In another preferred embodiment, the alkylphenol 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 combinations thereof.

[0021] In another preferred embodiment, the ionic surfactant is selected from the group consisting of CHAPS, sodium deoxycholate, or combinations thereof.

[0022] In another preferred embodiment, the bioactive perforating agent is selected from the group consisting of streptococcal hemolysin O, melitoxin, or combinations thereof.

[0023] In another preferred embodiment, the polar solvent is selected from the group consisting of DMSO, DMF, or combinations thereof.

[0024] In another preferred embodiment, the antibody comprises a primary antibody, or a combination of a primary antibody and a secondary antibody. Secondary antibody incubation may be performed as needed to enhance the primary antibody signal.

[0025] In another preferred embodiment, the concentration of the protein-nucleic acid fragment release agent is 0.01%-50% (v / v), more preferably 0.05%-25% (v / v).

[0026] In another preferred embodiment, the Tween 20 concentration is 0.01% - 5% (v / v), more preferably 0.05% - 0.1% (v / v).

[0027] In another preferred embodiment, the NP40 concentration is 0.01% - 5% (v / v), more preferably 0.05% - 0.1% (v / v).

[0028] In another preferred embodiment, the concentration of IGEPEAL CA-630 is 0.01% - 5% (v / v), more preferably 0.05% - 0.1% (v / v).

[0029] In another preferred embodiment, the CHAPS concentration is 0.01% - 5% (v / v), more preferably 0.05% - 0.1% (v / v).

[0030] In another preferred embodiment, the TritonX-100 concentration is 0.01% - 5% (v / v), more preferably 0.05% - 0.1% (v / v).

[0031] In another preferred embodiment, the concentration of sodium deoxycholate is 0.01% - 5% (v / v), more preferably 0.05% - 0.1% (v / v).

[0032] In another preferred embodiment, the concentration of the digitalis saponin is 0.01%-5% (v / v), more preferably 0.05%-0.1% (v / v).

[0033] In another preferred embodiment, the DMSO has a volume percentage of 0.5%-50% (v / v), more preferably 2.5%-25% (v / v).

[0034] In another preferred embodiment, the concentration of the streptococcal hemolysin O is 0.01-20 μg / mL.

[0035] In another preferred embodiment, the concentration of the melitin is 0.1-500 μM.

[0036] In another preferred embodiment, the volume percentage of the DMF is 0.5%-50% (v / v), more preferably 2.5%-25% (v / v).

[0037] In another preferred embodiment, in step (S6), the temperature T is 0~30°C, more preferably 4~20°C.

[0038] In another preferred embodiment, in step (S6), the time t is 20~240 min, more preferably 40~90 min.

[0039] In another preferred embodiment, the temperature T of the nucleic acid fragmentation process in 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 includes continuous reactions at different temperatures.

[0040] In another preferred embodiment, the temperature T mentioned in step (S6) during the activation and release process can be a fixed value or a combination of multiple temperature values. For example, T can be a fixed value of 18°C, meaning the activation and release temperature is 18°C ​​throughout the entire process, and the reaction lasts for one hour. T can also be multiple values, including 15°C, 18°C, and 20°C, meaning the activation and release temperature is divided into multiple segments: reacting at 15°C for 20 minutes, reacting at 18°C ​​for 20 minutes, and reacting at 20°C for 20 minutes.

[0041] In another preferred embodiment, the nucleic acid fragmentation process in step (S6) includes: a first activation-release reaction, a second activation-release reaction, a third activation-release reaction, or a combination thereof; wherein,

[0042] The temperature of the first activation and release reaction is 15°C, and the reaction time is 20 min;

[0043] The second activation and release reaction was carried out at a temperature of 18°C ​​for 20 minutes.

[0044] The temperature of the third activation and release reaction is 20°C, and the reaction time is 20 min.

[0045] In another preferred embodiment, the metal chelating agent is selected from the group consisting of EDTA, EGTA, or combinations thereof.

[0046] In another preferred embodiment, the concentration of the metal ion chelating agent is 1~50mM.

[0047] In another preferred embodiment, the metal chelating agent comprises EDTA at a concentration of 1-50 mM and EGTA at a concentration of 1-5 mM.

[0048] In another preferred embodiment, step (S6) includes any one of the following steps:

[0049] (S61) Under temperature T1 (°C), in the activation and release reaction system, the nuclease-antibody-cell or cell nucleus complex is treated with a protein-nucleic acid fragment release agent for time t1 (min) to perform nucleic acid fragmentation and release of nucleic acid fragments;

[0050] (S62) Under temperature T2 (°C), in the activation and release reaction system, the nuclease-antibody-cell complex is treated with a protein-nucleic acid fragment release agent for time t2 (min) to perform nucleic acid fragmentation and release nucleic acid fragments; a metal chelating agent is added, and the reaction is terminated and nucleic acid fragments are further released under temperature T3 (°C) for time t3 (min);

[0051] (S63) Under temperature T4 (°C), the nuclease-antibody-cell complex is treated in metal ion buffer (without protein-nucleic acid fragment release agent) for time t4 (min), and then the reaction process of step S61 is carried out.

[0052] (S64) At a temperature of T4 (°C), the nuclease-antibody-cell complex is treated in a metal ion buffer (without a protein-nucleic acid fragment release agent) for a time of t4 (min), followed by the reaction process described in step S62.

[0053] In another preferred embodiment, the temperature T1 is 0~30℃, more preferably 10~20℃.

[0054] In another preferred embodiment, the time t1 is 20~240 min, more preferably 40~60 min.

[0055] In another preferred embodiment, the temperature T2 is 0~30℃, more preferably 4~10℃.

[0056] In another preferred embodiment, the time t2 is 20~240 min, more preferably 20~40 min.

[0057] In another preferred embodiment, the temperature T3 is 0~30℃, more preferably 10~20℃.

[0058] In another preferred embodiment, the time t3 is 20~240 min, more preferably 20~50 min.

[0059] In another preferred embodiment, the temperature T4 is 0~30℃, more preferably 0~10℃.

[0060] In another preferred embodiment, the time t4 is 1~240 min, more preferably 20~60 min.

[0061] In another preferred embodiment, the temperatures T1 to T4 can be the same temperature throughout the entire process, or they can be a combination of multiple temperature stages.

[0062] In another preferred embodiment, in step (S6), the activation and release reaction system includes: a protein-nucleic acid fragment complex release agent and a metal ion buffer with a concentration of 0.01 mM to 10 mM.

[0063] In another preferred embodiment, 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 combinations thereof.

[0064] In another preferred embodiment, the concentration of metal ions in the metal ion buffer is 0.01 mM to 10 mM.

[0065] In another preferred embodiment, the metal ion buffer solution comprises a CaCl2 solution with a concentration of 0.01 mM to 10 mM.

[0066] In another preferred embodiment, in step (S6), the activation and release reaction system comprises: 0.01%-0.5% (v / v) of digitalis saponins, 0.5%-50% (v / v) of DMSO, and a CaCl2 solution with a concentration of 0.01 mM to 10 mM.

[0067] In another preferred embodiment, in step (S6), the pH of the activation release reaction system is 6.0-8.5.

[0068] In another preferred embodiment, in step (S6), the activation release reaction system further includes a permeation buffer.

[0069] In another preferred embodiment, the permeation buffer comprises PB, Tris, and HEPES, or a combination thereof.

[0070] In another preferred embodiment, the pH of the permeation buffer is 7.0 to 7.8; more preferably 7.2 to 7.6.

[0071] In another preferred embodiment, the permeation buffer is a salt containing 1-500 mM NaCl or other salts with equivalent conductivity.

[0072] In another preferred embodiment, in (S1), the cell comprises a eukaryotic cell, preferably an animal or plant cell, and more preferably a mammalian cell.

[0073] In another preferred embodiment, the mammalian cells are selected from the group consisting of hamster cells, human cells, or mouse cells.

[0074] In another preferred embodiment, in (S1), the cell density in the cell sample is 1 × 10⁻⁶. 3 ~5×10 6 per mL.

[0075] In another preferred embodiment, in (S1), the cell nucleus sample is prepared with a cell density of 1×10⁻⁶ cells. 3 ~5×10 6 Cell nucleus samples after cell lysis buffer treatment of cells per mL.

[0076] In another preferred embodiment, (S2) the washing includes: adding cell washing buffer, centrifuging to remove the supernatant, obtaining a cell or cell nucleus sample, and then resuspending the cell or cell nucleus sample with cell washing buffer.

[0077] 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 the remainder being water.

[0078] In another preferred embodiment, the magnetic beads are concanavalin A magnetic beads, hereinafter referred to as ConA magnetic beads.

[0079] In another preferred embodiment, the pretreatment process of the pretreated magnetic beads includes the following steps: treating the ConA magnetic bead solution with a binding buffer, placing it on a magnetic rack and letting it stand until the solution is clear, discarding the supernatant, and obtaining the pretreated magnetic beads.

[0080] In another preferred embodiment, in step (S4), the incubation time of the antibody is 10 min to 20 hours.

[0081] In another preferred embodiment, step (S4) further includes using a secondary antibody to bind the antibody.

[0082] In another preferred embodiment, in (S5), the nuclease is selected from the group consisting of pAG-DNase I, pAG-MNase, pAG-Benzonase, or combinations thereof.

[0083] In another preferred embodiment, the DNase I in the pAG-DNase I is cloned from bovine (… Bos taurus ).

[0084] In another preferred embodiment, the MNase in the pAG-MNase is cloned from Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus ).

[0085] In another preferred embodiment, the pAG-Benzonase contains a Benzonase clone of Serratia marcescens (Serratia marcescens). Serratia marcescens ).

[0086] In another preferred embodiment, in step (S5), the nuclease is fused with a domain or polypeptide that can bind to antibodies, thereby targeting and immobilizing the nuclease near the target protein.

[0087] In another preferred embodiment, the antibody-binding domain or polypeptide is selected from the group consisting of protein A, protein G, protein L, or combinations thereof.

[0088] In another preferred embodiment, the antibody-binding domain or polypeptide is selected from antibodies that can specifically recognize the FC region of the antibody; the nuclease cleaves nucleic acid near the target protein through antibody targeting and immobilization to form a free nucleic acid-protein complex.

[0089] In another preferred embodiment, the nuclease possesses endonuclease activity, is independent of a specific sequence, can cleave single-stranded or double-stranded nucleic acids, and has metal ion activation characteristics.

[0090] In another preferred embodiment, the nuclease includes, but is not limited to, pAG-DNase I, pAG-MNase, or pAG-Benzonase.

[0091] In another preferred embodiment, the nuclease possesses metal ion activation characteristics.

[0092] In another preferred embodiment, step (S5) further includes the following step: after the treatment, washing the nuclease with a detergent for a washing time of 1 to 60 minutes.

[0093] In another preferred embodiment, step (S7) includes: centrifuging the released nucleic acid fragments to obtain the supernatant, and adding DNA extraction reagent to the supernatant for incubation.

[0094] In another preferred embodiment, in step (S7), the DNA extraction reagent comprises: 2-10 μL 0.5M EDTA; 1-8 μL 10% SDS; and 0.2-5 μL 20 mg / mL proteinase K.

[0095] In a second aspect of the present invention, a kit for constructing a nucleic acid library using the method described in the first aspect of the present invention is provided, comprising:

[0096] (1) Protein-nucleic acid fragment release promoters;

[0097] (2) Metal ion buffer solution;

[0098] (3) Cell washing buffer; and

[0099] (4) Fragmentation reaction reagents.

[0100] In another preferred embodiment, the protein-nucleic acid fragment release agent is selected from the group consisting of: nonionic surfactants, ionic surfactants, bioactive pore-forming agents, polar solvents, or combinations thereof.

[0101] In another preferred embodiment, the nonionic surfactant is selected from the group consisting of polysorbate compounds, alkylphenol polyoxyethylene ether compounds, NP40 or IGEPA CA-630, digitalis saponins, or combinations thereof.

[0102] 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 combinations thereof.

[0103] In another preferred embodiment, the alkylphenol 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 combinations thereof.

[0104] In another preferred embodiment, the ionic surfactant is selected from the group consisting of CHAPS, sodium deoxycholate, or combinations thereof.

[0105] In another preferred embodiment, the bioactive perforating agent is selected from the group consisting of streptococcal hemolysin O, melitoxin, or combinations thereof.

[0106] In another preferred embodiment, the polar solvent is selected from the group consisting of DMSO, DMF, or combinations thereof.

[0107] In another preferred embodiment, the protein-nucleic acid fragment release agent is selected from the group consisting of DMSO, SMF, Tween-20, NP40, Triton X-100, CHAPS, sodium deoxycholate, streptococcal hemolysin O, melitrix venom peptide, or combinations thereof.

[0108] In another preferred embodiment, the DMSO has a volume percentage of 0.5%-50% (v / v), more preferably 2.5%-25% (v / v).

[0109] In another preferred embodiment, the concentration of the streptococcal hemolysin O is 0.01-20 μg / mL.

[0110] In another preferred embodiment, the concentration of the melitin is 0.1-500 μM.

[0111] In another preferred embodiment, the volume percentage of the DMF is 0.5%-50% (v / v), more preferably 2.5%-25% (v / v).

[0112] In another preferred embodiment, 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 combinations thereof.

[0113] In another preferred embodiment, the concentration of metal ions in the metal ion buffer is 0.01 mM to 10 mM.

[0114] In another preferred embodiment, the metal ion buffer solution comprises a CaCl2 solution with a concentration of 0.01 mM to 10 mM.

[0115] In another preferred embodiment, the kit also includes a metal chelating agent.

[0116] In another preferred embodiment, the metal chelating agent is selected from the group consisting of EDTA, EGTA, or combinations thereof.

[0117] In another preferred embodiment, the concentration of the metal ion chelating agent is 1~50mM.

[0118] In another preferred embodiment, the metal chelating agent comprises EDTA at a concentration of 1-50 mM and EGTA at a concentration of 1-5 mM.

[0119] In another preferred embodiment, the kit also includes a permeation buffer.

[0120] In another preferred embodiment, the permeation buffer includes PB, Tris, and HEPES buffers.

[0121] In another preferred embodiment, the pH of the permeation buffer is 7.0 to 7.8; more preferably 7.2 to 7.6.

[0122] In another preferred embodiment, the permeation buffer contains 50-150 mM NaCl or other salts with equivalent conductivity.

[0123] In another preferred embodiment, the permeation buffer further contains a cell protein protectant, which is 1-100 mM Glycine, 0.1%-2% (v / v) BSA, or other similar substances.

[0124] In another preferred embodiment, the permeation buffer comprises a detergent selected from the group consisting of: digitalis saponins, Tween 20, SDS, NP40, CHAPS, sodium deoxycholate, or combinations thereof; preferably digitalis saponins, Tween 20, SDS, or NP40.

[0125] In another preferred embodiment, the pH of the cell washing buffer is 7.2 to 7.6.

[0126] In another preferred embodiment, the cell washing buffer contains 100-150 mM NaCl or other salts with equivalent conductivity.

[0127] In another preferred embodiment, the fragmentation reaction reagent includes, but is not limited to, nucleases, magnetic beads, primary antibodies, and secondary antibodies.

[0128] In another preferred embodiment, the kit further includes DNA extraction reagents, reaction termination reagents, or reagents for PCR amplification.

[0129] In another preferred embodiment, the DNA extraction reagent includes EDTA, SDS, and proteinase K.

[0130] In a third aspect of the invention, a method for measuring protein-nucleic acid interactions is provided, comprising:

[0131] (S1) Constructing a nucleic acid library using the method described in the first aspect of the present invention; and

[0132] (S2) Sequencing of the nucleic acid library to generate multiple sequencing reads for measuring protein-nucleic acid interactions.

[0133] In another preferred embodiment, the protein includes a DNA-binding protein with weak DNA binding affinity or with highly dynamic DNA binding affinity.

[0134] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0135] Figure 1 The diagram shows a comparison between the traditional CUT&RUN experimental procedure and the CUT&RUNLT procedure of this invention.

[0136] Figure 2 The graphs show the IGV peaks of the Pol-II antibody group with and without DMSO addition, with the upper and lower graphs representing IGV peaks at genomic coordinate scales of 50kb and 20kb, respectively.

[0137] Figure 3 The image shows the IGV peaks of the CTCF antibody group with and without DMSO addition. The bottom row shows the control peaks of ChIP-seq. The genomic coordinate scale is 50kb.

[0138] Figure 4 The image shows the IGV peaks of the H3K4me3 antibody group with and without DMSO addition. The bottom row shows the control peaks from ChIP-seq. The genomic coordinate scale is 50kb.

[0139] Figure 5 The IGV peak diagrams of Pol-II antibody with different DMSO groups at different activation temperatures are shown. In A, the activation temperature is 4℃, and in B, the activation temperature is 20℃.

[0140] Figure 6 The IGV peak diagrams of the Pol-II antibody group at different activation temperatures are shown.

[0141] Figure 7 The results of IGV peaks in the Pol-II antibody group using different cut & run methods are shown.

[0142] Figure 8 The results of IGV peaks in the CTCF antibody group using different cut & run methods are shown.

[0143] Figure 9 The IGV peak diagrams for different antibody groups with different nucleases and different DMF additions are shown. In this diagram, A represents the Pol-II antibody group and B represents the CTCF antibody group.

[0144] Figure 10 The image shows the IGV peaks of cell nuclear samples with and without the addition of DMSO in different antibody groups. A represents the Pol-II antibody group, B represents the CTCF antibody group, and C represents the H3K4me3 antibody group. The bottom row of B and C represents the control peaks from ChIP-seq.

[0145] Figure 11 The image shows the IGV peaks of the Pol-II antibody group after adding different concentrations of protein-nucleic acid fragment release agents.

[0146] Figure 12 The image shows the IGV peaks of the Pol-II antibody group after adding different concentrations of protein-nucleic acid fragment release agent, where SLO is the abbreviation for streptococcal hemolysin O and MLT is the abbreviation for melitrix venom peptide.

[0147] Figure 13 The image shows the IGV peaks of the Pol-II antibody group after adding different combinations of protein-nucleic acid fragment release agents.

[0148] Figure 14 The diagram shows the IGV peaks of the Pol-II antibody and CTCF antibody in a one-step multi-temperature gradient activation system, with the order in which the protein-nucleic acid fragment release agent was added to the activation system. Detailed Implementation

[0149] Through in-depth research and extensive experimental screening, the inventors unexpectedly discovered a CUT&RUNLT method. By altering the enzyme digestion and nucleic acid release conditions of traditional CUT&RUN (0-4℃ low-temperature enzyme digestion of chromatin DNA + 37℃ high-temperature release of the digested fragments), and by adding a cell-stimulating protein-nucleic acid fragment release agent to the enzyme digestion system and employing a low-temperature release scheme, the experimental quality can be significantly improved. The signal-to-noise ratio and sensitivity of the experimental results are far superior to traditional methods, and some experimental results are even superior to CUT&Tag technology. Furthermore, the results for testing various nucleases are superior to those of traditional CUT&RUN. Based on this, the present invention was completed.

[0150] the term

[0151] 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 invention pertains.

[0152] As used herein, “including” or “containing” includes “comprising,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0153] As used herein, "CUT&RUNLT method of the present invention", "CUT&RUNLT process of the present invention", "one-step CUT&RUNLT method of the present invention", and "two-step CUT&RUNLT method of the present invention" can be used interchangeably and all refer to the method of the first aspect of the present invention. Specifically, by changing the enzyme digestion and nucleic acid release conditions of traditional CUT&RUN (low temperature enzyme digestion of chromatin DNA at 0-4℃ + high temperature release of enzyme-digested fragments at 37℃), a method of adding a protein-nucleic acid fragment release agent to the enzyme digestion system and using low temperature to release nucleic acid fragments is adopted.

[0154] As used herein, “protein nucleic acid fragment release agent,” “cell permeability agent,” and “release agent” are interchangeable and all refer to reagents used in the methods described in this invention that can promote the release of nucleic acid fragments or their complexes formed by binding with antibodies.

[0155] The main advantages of this invention include:

[0156] (1) Compared with the traditional CUT&RUN method, the CUT&RUNLT method of the present invention significantly improves the experimental quality and significantly increases the number of peaks.

[0157] (2) The CUT&RUNLT method of the present invention can release nucleic acid fragments that cannot be released in the traditional CUT&RUN method, thus expanding the range of detection targets.

[0158] (3) The signal-to-noise ratio and sensitivity of the experimental results data of the CUT&RUNLT method of the present invention are significantly better than those of the traditional method. Some experimental results are even better than those of the CUT&Tag technology, and the results of testing a variety of nucleases are better than those of the traditional CUT&RUN method.

[0159] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, all experimental materials and reagents involved in this invention are commercially available.

[0160] Experimental reagents

[0161] This embodiment used concanavalin A magnetic beads (NovoNGS® concanavalin A coated magnetic beads: N251), DNA extraction magnetic beads (tagged DNA extraction magnetic beads: N245, NovoNGS® DNA purification magnetic beads: N240), and library construction kits (NovoNGS® Illumina® sequencing platform DNA library preparation kit: N256; NovoNGS® Illumina® sequencing platform multiplex indexed primer set 3: E163) from Suzhou Nearshore Protein Technology Co., Ltd. The primary antibodies used in this embodiment were 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 this invention are human 293F cells. The self-prepared reagent formulations for each buffer used in the experiment are shown in Table 1 below: (all formulations are based on 1 mL volume; volumes can be scaled up proportionally).

[0162] Table 1

[0163]

[0164] Experimental methods:

[0165] (1) Traditional CUT & RUN process

[0166] The conventional CUT&RUN process in the prior art (cited in Skene, P., Henikoff, J. & Henikoff, S. Nat Protoc 13, 1006–1019 (2018), doi:10.1038 / nprot.2018.015) includes the following steps:

[0167] 1. Cell preparation

[0168] 1-1) Collect and count fresh cells at room temperature. Take the required cells into a new 1.5 mL centrifuge tube, centrifuge at 600g for 5 min at room temperature, carefully discard the culture medium supernatant, and obtain the cell pellet.

[0169] 1-2) Add 1 mL of washing buffer to the cell pellet from step 1-1), gently pipette to mix, centrifuge at 600 g for 5 min at room temperature, and carefully discard the supernatant;

[0170] 1-3) Add 90 μL of washing buffer to the cell pellet from step 1-2) to resuspend the cells and obtain a cell sample.

[0171] 2. Preparation and cell binding of ConA magnetic beads

[0172] 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 1.5mL centrifuge tube, place it on a magnetic rack and let it stand for 2 min until the solution is clear, then discard the supernatant;

[0173] 2-2) Add 100 μL of binding buffer and mix by pipetting.

[0174] 2-3) Place on a magnetic rack and let stand for 2 minutes until the solution is clear, then discard the supernatant;

[0175] 2-4) Repeat steps 2-2) and 2-3) once;

[0176] 2-5) Remove the centrifuge tube, add 10 μL of binding buffer, mix well by pipetting, and place on ice for later use;

[0177] 2-6) Add 10 μL of pretreated ConA magnetic beads to the 90 μL cell sample from step 1-3), gently mix by pipetting, and place on a rotary mixer at room temperature for 10 min to obtain the ConA magnetic bead-cell mixture.

[0178] 3. ConA magnetic beads - cell binding to primary antibody

[0179] 3-1) Dilute the primary antibody with antibody buffer. Each experimental sample requires 50 μL of primary antibody dilution solution. The dilution ratio of the primary antibody should be based on the instructions. Place the prepared primary antibody dilution solution on ice for later use.

[0180] 3-2) Place the centrifuge tube containing the ConA magnetic bead-cell mixture from step 2-6) on a magnetic rack and let it stand for 2 min. After the magnetic beads are completely separated from the liquid, carefully discard the supernatant, remove the centrifuge tube, add 50 μL of primary antibody dilution buffer, and incubate at room temperature for 2 h or at 4°C overnight.

[0181] 4. ConA magnetic beads - cell washing

[0182] 4-1) Remove the centrifuge tube from step 3-2) after incubating the primary antibody, place it on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely separated from the liquid, carefully discard the supernatant.

[0183] 4-2) Remove the centrifuge tube from the magnetic rack, add 1 mL of Dig-washing buffer, resuspend the ConA magnetic bead-cell mixture, gently pipette to mix, and let stand at room temperature for 2 min;

[0184] 4-3) Place it on a magnetic rack and let it stand for 2 minutes until the magnetic beads are completely separated from the liquid. Then carefully discard the supernatant.

[0185] 4-4) Repeat steps 4-2) and 4-3) twice.

[0186] 5. Binding and washing of pAG-MNase

[0187] 5-1) Remove the centrifuge tube from the magnetic rack, add 50 μL of Dig-washing buffer and pAG-MNase to a final concentration of 200 ng / mL, gently pipette to resuspend the ConA magnetic bead-cell mixture, and incubate at 4°C for 1 h.

[0188] 5-2) Place the centrifuge tube incubated with pAG-MNase on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely separated from the liquid, carefully discard the supernatant.

[0189] 5-3) Remove the centrifuge tube from the magnetic rack, add 1 mL of Dig-washing buffer, resuspend the ConA magnetic bead-cell mixture, gently pipette to mix, and let stand at room temperature for 2 min;

[0190] 5-4) Place it on a magnetic rack and let it stand for 2 minutes until the magnetic beads are completely separated from the liquid. Then carefully discard the supernatant.

[0191] 5-5) Repeat steps 5-3) and 5-4) twice.

[0192] 6. Fragmentation reaction

[0193] 6-1) Remove the centrifuge tube from the magnetic rack, add 150 μL of Dig-washing buffer, gently pipette to mix the cells, and place on ice for later use.

[0194] 6-2) Add 3 μL of 100 mM CaCl2 to the centrifuge tube from step 6-1), mix gently, and immediately place on ice for 30 min.

[0195] 7. Chromatin fragment release

[0196] 7-1) Add 100 μL of 2× termination buffer to the centrifuged mixture after the reaction in step 6-2), mix by pipetting or vortexing, and incubate in a metal bath or water bath at 37°C for 10 min to release chromatin fragments into the supernatant.

[0197] 7-2) Centrifuge at 16000 g for 5 min at 4℃, place the centrifuge tube on a magnetic rack and let it stand for 2 min. After the magnetic beads are completely separated from the liquid, carefully transfer the supernatant to a new centrifuge tube to obtain the fragmented nucleic acid.

[0198] 8. DNA extraction

[0199] 8-1) Add 2 volumes of tagged DNA extraction magnetic beads (Novoprotein, catalog number N245) equilibrated to room temperature to the supernatant of step 7-2), gently pipette to mix, and let stand at room temperature for 5 min.

[0200] 8-2) Transfer the PCR tube to a magnetic rack and let it stand for 5 minutes to separate the magnetic beads from the liquid. Carefully discard the supernatant.

[0201] 8-3) Add 200 μL of freshly prepared 80% ethanol to the PCR tube, let it stand at room temperature for 1 min, then carefully discard the supernatant, and keep the PCR tube on the magnetic rack.

[0202] 8-4) Repeat step 8-3) once;

[0203] 8-5) Place the PCR tube on a magnetic rack, open the PCR tube cap, and air dry at room temperature (about 4 minutes) to allow the ethanol to evaporate completely;

[0204] 8-6) Remove the PCR tubes from the magnetic rack, add 22 μL of ddH2O to each tube, mix by pipetting, and let stand at room temperature for 3 min.

[0205] 8-7) Place the PCR tube on a magnetic rack and let it stand for 2 minutes to completely separate the magnetic beads from the liquid. Carefully aspirate 20 μL of the supernatant into a new PCR tube, which is the purified protein-interacting nucleic acid fragment.

[0206] 9. Library Construction

[0207] Use the DNA Library Preparation Kit (catalog number N256) on the NovoNGS® Illumina® Sequencing Platform; construct sequencing libraries from the purified product using the NovoNGS® Illumina® Sequencing Platform with multiplex indexed primer set 3 (catalog number E163), following the instructions in the manufacturer's manual.

[0208] 10. Library sequencing

[0209] After purification, the sequencing library products were sent to a sequencing company for sequencing. The sequencing results were analyzed on the Galaxy website (https: / / usegalaxy.org / ), and the target protein binding maps were viewed using IGV software.

[0210] (2) The CUT & RUNLT process of the present invention:

[0211] like Figure 1 As shown, compared with the traditional CUT&RUN procedure, this invention changes the enzyme digestion and nucleic acid release conditions of the traditional CUT&RUN (0-4℃ low-temperature enzyme digestion of chromatin DNA + 37℃ high-temperature release of the digested fragments). By adding a protein-nucleic acid fragment release agent to the enzyme digestion system and adopting a low-temperature release scheme for nucleic acid fragments, the experimental quality can be significantly improved. The signal-to-noise ratio and sensitivity of the experimental results are far superior to those of the traditional method. The CUT&RUNLT procedure of this invention includes the following steps:

[0212] 1. Cell preparation

[0213] 1-1) Collect and count fresh cells at room temperature. Take the required cells into a new 1.5 mL centrifuge tube, centrifuge at 600 g for 5 min at room temperature, carefully discard the culture medium supernatant, and obtain the cell pellet.

[0214] 1-2) Add 1 mL of washing buffer to the cell pellet from step 1-1), gently pipette to mix, centrifuge at 600 g for 5 min at room temperature, and carefully discard the supernatant.

[0215] 1-3) Add 90 μL of washing buffer to the cell pellet from step 1-2) to resuspend the cells and obtain a cell sample.

[0216] 2. Preparation and cell binding of ConA magnetic beads

[0217] 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 rack and let it stand for 2 min until the solution is clear, then discard the supernatant;

[0218] 2-2) Add 100 μL of binding buffer and mix by pipetting.

[0219] 2-3) Place on a magnetic rack and let stand for 2 minutes until the solution is clear, then discard the supernatant;

[0220] 2-4) Repeat step 2-3) once;

[0221] 2-5) Remove the centrifuge tube, add 10 μL of binding buffer, mix well by pipetting, and place on ice for later use;

[0222] 2-6) Add 90 μL of the cell sample resuspended in steps 1-3) to 10 μL of the prepared ConA magnetic beads, gently pipette to mix, and place on a rotary mixer at room temperature for 10 min to obtain the ConA magnetic bead-cell mixture.

[0223] 3. ConA magnetic beads - cell binding to primary antibody

[0224] 3-1) Dilute the primary antibody with antibody buffer. Each experimental sample requires 50 μL of primary antibody dilution solution. The dilution ratio of the primary antibody should be based on the instructions. Place the prepared primary antibody dilution solution on ice for later use.

[0225] 3-2) Place the centrifuge tube containing the ConA magnetic bead-cell mixture from step 2-6) on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely separated from the liquid, carefully discard the supernatant, remove the centrifuge tube, add 50 μL of primary antibody dilution buffer, and incubate at room temperature for 2 hours or at 4°C overnight.

[0226] 4. ConA magnetic beads - cell washing

[0227] 4-1) Remove the centrifuge tube from step 3-2) after incubating the primary antibody, place it on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely separated from the liquid, carefully discard the supernatant.

[0228] 4-2) Remove the centrifuge tube from the magnetic rack, add 200 μL of Dig-washing buffer, resuspend the ConA magnetic bead-cell mixture, gently pipette to mix, and let stand at room temperature for 2 min;

[0229] 4-3) Place it on a magnetic rack and let it stand for 2 minutes until the magnetic beads are completely separated from the liquid. Then carefully discard the supernatant.

[0230] 4-4) Repeat steps 4-2) and 4-3) once.

[0231] 5. Binding and washing of pAG-MNase

[0232] 5-1) Remove the centrifuge tube from the magnetic rack, add 50 μL of Dig-washing buffer and pAG-MNase to a final concentration of 200 ng / mL, gently pipette to resuspend the ConA magnetic bead-cell mixture, and incubate at 4°C for 1 h.

[0233] 5-2) Place the centrifuge tube incubated with pAG-MNase on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely separated from the liquid, carefully discard the supernatant.

[0234] 5-3) Remove the centrifuge tube from the magnetic rack, add 200 μL of Dig-washing buffer, resuspend the ConA magnetic bead-cell mixture, gently pipette to mix, and let stand at room temperature for 2 min;

[0235] 5-4) Place it on a magnetic rack and let it stand for 2 minutes until the magnetic beads are completely separated from the liquid. Then carefully discard the supernatant.

[0236] 5-5) Repeat steps 5-3) and 5-4) once.

[0237] 6. Fragmentation reaction and chromatin release

[0238] 6-1) Remove the centrifuge tube from the magnetic rack, add 50 μL of activation buffer, gently pipette to mix the cells, and then perform fragmentation and chromatin release using the following two methods, which will be referred to as the one-step method and the two-step method from now on.

[0239] (1) Option 1: The one-step method includes the following steps:

[0240] Place in an 18°C ​​metal bath or PCR instrument and incubate for 60 min to release nucleic acids during fragmentation.

[0241] (2) Option 2: The two-step method includes the following steps:

[0242] Place in a 4℃ metal bath or PCR instrument and incubate for 40 min; after incubation, add 10 mM EDTA and 2 mM EGTA to the reaction system and incubate at 18℃ for 50 min; terminate the reaction and release nucleic acids.

[0243] 6-2) Place the centrifuge tube from step 6-1) on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely separated from the liquid, carefully transfer the supernatant to a new PCR tube to obtain the fragmented nucleic acid.

[0244] 7. DNA extraction

[0245] 7-1) Add 5 μL of 0.5M EDTA, 4 μL of 10% SDS, and 1 μL of 20 mg / mL proteinase K to the supernatant obtained in step 6-2), mix well by pipetting, and let it react at room temperature for 5 min.

[0246] 7-2) Nucleic acid fragments were purified and recovered using magnetic beads. Two volumes of tagged DNA extraction magnetic beads equilibrated to room temperature were added to the system from the previous step. The mixture was gently blown to mix and then allowed to stand at room temperature for 5 min.

[0247] 7-3) Transfer the PCR tube to a magnetic rack and let it stand for 5 minutes to separate the magnetic beads from the liquid. Carefully discard the supernatant.

[0248] 7-4) Add 200 μL of freshly prepared 80% ethanol to the PCR tube, let it stand at room temperature for 1 min, then carefully discard the supernatant, and keep the PCR tube on the magnetic rack.

[0249] 7-5) Repeat step 7-4) once;

[0250] 7-6) Place the PCR tube on a magnetic rack, open the PCR tube cap, and air dry at room temperature (about 4 minutes) to allow the ethanol to evaporate completely;

[0251] 7-7) Remove the PCR tubes from the magnetic rack, add 22 μL of ddH2O to each tube, mix by pipetting, and let stand at room temperature for 3 min.

[0252] 7-8) Place the PCR tube on a magnetic rack and let it stand for 2 minutes to completely separate the magnetic beads from the liquid. Carefully aspirate 20 μL of supernatant into a new PCR tube. The sample can be stored at -20℃ or directly proceeded to the next step of library construction.

[0253] 8. Library Construction

[0254] Use the DNA Library Preparation Kit (catalog number N256) on the NovoNGS® Illumina® Sequencing Platform; construct sequencing libraries from the purified product using the NovoNGS® Illumina® Sequencing Platform with multiplex indexed primer set 3 (catalog number E163), following the instructions in the manufacturer's manual.

[0255] 9. Library sequencing

[0256] After purification, the sequencing library products were sent to a sequencing company for sequencing. The sequencing results were analyzed on the Galaxy website (https: / / usegalaxy.org / ), and the target protein binding maps were viewed using IGV software.

[0257] Example 1: The effect of adding DMSO on the results of CUT & RUN experiments

[0258] This embodiment examines the effect of adding DMSO on the results of the cut & run experiment. The specific experimental grouping and experimental procedure design are shown in Table 2 below:

[0259] Table 2

[0260]

[0261] Experimental Procedure: The following experiments were conducted according to the one-step CUT&RUNLT experimental procedure of this invention. For detailed operation, please refer to the CUT&RUNLT procedure description of this invention in the experimental method above. The following is a brief description of the steps:

[0262] 1. Cell preparation

[0263] 1-1) Take 800,000 freshly harvested 293F cells, divide them into 8 equal parts, place them in 1.5 mL centrifuge tubes, 100,000 cells / sample, centrifuge at 600 g for 5 min at room temperature, carefully discard the supernatant, and obtain cell pellet;

[0264] 1-2) Add 1 mL of washing buffer to the cell pellet from step 1-1), gently pipette to mix, centrifuge at 600 g for 5 min at room temperature, carefully discard the supernatant to obtain the cell pellet;

[0265] 1-3) Add 90 μL of washing buffer to the cell pellet from step 1-2) to resuspend the cells.

[0266] 2. Preparation and cell binding of ConA magnetic beads

[0267] 3. ConA magnetic beads - cell binding to primary antibody

[0268] 4. ConA magnetic beads - cell washing

[0269] 5. Binding and washing of pAG-MNase

[0270] 6. Fragmentation reaction and chromatin release

[0271] 6-1) The activation systems in groups 1, 3, 5 and 7 of Table 2 above include: 50 μL washing buffer, DMSO with a final concentration of 10% and CaCl2 with a final concentration of 1mM.

[0272] The activation systems in groups 2, 4, 6 and 8 consist of: 50 μL of wash buffer, 1 mM CaCl2, and no DMSO added.

[0273] After gently mixing the cells by pipetting, incubate them at 18°C ​​for 60 min on a PCR instrument to release nucleic acids during fragmentation.

[0274] 6-2) Place the reaction centrifuge tube from step 6-1) on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely separated from the liquid, carefully transfer the supernatant to a new PCR tube to obtain the fragmented nucleic acid.

[0275] 7. DNA extraction

[0276] 8. Library Construction

[0277] The same number of cycles was used for library amplification, with groups 1 to 8 above amplifying for 14 cycles each.

[0278] 9. Library sequencing

[0279] The purified library products were sent to a sequencing company for sequencing. Sequencing results were analyzed on the Galaxy website (https: / / usegalaxy.org / ). Target protein binding maps were viewed using IGV software. Sequencing result analysis is as follows: Figure 2-4 As shown in Table 3, the effect of adding or not adding DMSO on the number of library peaks in different antibody groups is shown in Table 3.

[0280] Table 3

[0281]

[0282] The results showed that, as indicated in Table 3, the number of peaks increased significantly after adding 10% DMSO. The peak diagram on IGV showed that, as a negative control, the addition of DMSO did not affect the results in the IgG group. However, in the three antibody groups (Pol-II, CTCF, and H3Kme3), the groups without DMSO showed some peak loss. This indicates that the addition of DMSO enhanced previously existing peak signals, and effectively detected previously undetectable peak signals. Figures 2-4 (As indicated by the arrows in the diagram). Furthermore, compared to the antibody group without DMSO, the number of peaks with added DMSO in Pol-II increased by more than 2.6 times; the number of peaks with added DMSO in CTCF increased by approximately 56%; and the number of peaks with added DMSO in H3Kme3 increased by approximately 29%. Therefore, it can be inferred that adding DMSO to a final concentration of 10% during the activation step can promote the release of targeted cleaved nucleic acid fragments into the extracellular supernatant.

[0283] Example 2: Effect of different concentrations of DMSO added on test results

[0284] This embodiment examines the effect of adding different concentrations of DMSO on the test results. The specific experimental group design is shown in Table 4 below:

[0285] Table 4

[0286]

[0287] Experimental Procedure: The following experiments were conducted according to the one-step CUT&RUNLT experimental procedure of this invention. For detailed operation, please refer to the CUT&RUNLT procedure description of this invention in the experimental method above. The following is a brief description of the steps:

[0288] 1. Cell preparation

[0289] 1-1) Take 1.8 million freshly harvested 293F cells, divide them into 18 equal parts, place them 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;

[0290] 1-2) Add 1 mL of washing buffer to the cell pellet from step 1-1), gently pipette to mix, centrifuge at 600 g for 5 min at room temperature, carefully discard the supernatant to obtain the cell pellet;

[0291] 1-3) Add 90 μL of washing buffer to the cell pellet from step 1-2) to resuspend the cells.

[0292] 2. Preparation of ConA magnetic beads and binding with cells to obtain ConA magnetic bead-cell mixture.

[0293] 3. ConA magnetic beads - cell binding to primary antibody.

[0294] 1) Dilute the Pol-II primary antibody with antibody buffer. Each experimental sample requires 50 μL of primary antibody dilution solution. Keep on ice for later use.

[0295] 2) Place the centrifuge tube containing the ConA magnetic bead-cell mixture from step 2 on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely separated from the liquid, carefully discard the supernatant, remove the centrifuge tube, add 50 μL of Pol-II primary antibody dilution solution, and incubate at room temperature for 2 hours by rotation.

[0296] 4. ConA magnetic beads - cell washing

[0297] 5. Binding and washing of pAG-MNase

[0298] 6. Fragmentation reaction and chromatin release

[0299] 1) The activation systems for groups 1 and 10 in Table 4 above include: 50 μL of washing buffer and CaCl2 at a final concentration of 1 mM, without the addition of DMSO;

[0300] The activation systems for groups 2 and 11 consisted of 50 μL of wash buffer, 0.3% DMSO, and 1 mM CaCl2.

[0301] The activation systems for groups 3 and 12 consisted of 50 μL of wash buffer, 2.5% DMSO, and 1 mM CaCl2.

[0302] The activation systems for groups 4 and 13 consisted of 50 μL of wash buffer, 5% DMSO, and 1 mM CaCl2.

[0303] The activation systems for groups 5 and 14 consisted of 50 μL of wash buffer, 10% DMSO, and 1 mM CaCl2.

[0304] The activation systems for groups 6 and 15 consisted of 50 μL of wash buffer, 15% DMSO, and 1 mM CaCl2.

[0305] The activation systems for groups 7 and 16 consisted of 50 μL of wash buffer, 20% DMSO, and 1 mM CaCl2.

[0306] The activation systems for groups 8 and 17 consisted of 50 μL of wash buffer, 25% DMSO, and 1 mM CaCl2.

[0307] The activation systems for groups 9 and 18 consisted of 50 μL of wash buffer, 30% DMSO, and 1 mM CaCl2.

[0308] 2) After gently mixing the cells by pipetting, groups 1-9 were incubated at 4°C for 60 min on a PCR instrument to release nucleic acids while fragmenting; groups 10-18 were incubated at 20°C for 60 min on a PCR instrument to release nucleic acids while fragmenting.

[0309] 3) Place the centrifuge tube from the previous step on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely separated from the liquid, carefully transfer the supernatant to a new PCR tube to obtain the fragmented nucleic acid.

[0310] 7. DNA extraction

[0311] 8. Library Construction

[0312] The same number of cycles was used for library amplification; groups 1 to 18 above were all amplified for 14 cycles.

[0313] 9. Library sequencing

[0314] The purified library products were sent to a sequencing company for sequencing. Sequencing results were analyzed on the Galaxy website (https: / / usegalaxy.org / ). Target protein binding maps were viewed using IGV software. Sequencing result analysis is shown in Table 5. Figure 5 A- Figure 5 As shown in B.

[0315] Table 5

[0316]

[0317] The results showed that, at two different temperatures (4℃ and 20℃), with the same sequencing volume (1G), the number of peaks in the experimental groups increased significantly after adding DMSO at a final concentration of 0.3%–30% compared to the group without DMSO (Table 5). Furthermore, in the IGV peak diagram, it was observed that, compared to the group without DMSO, the peak signal was enhanced, the background was reduced, and the signal-to-noise ratio was more than doubled after adding DMSO. Figure 5 A- Figure 5 B). The results show that the release of nucleic acid fragments can be enhanced within the final concentration range of 0.3% to 30% of DMSO.

[0318] Example 3: The effect of different temperatures during the activation step on the test

[0319] This embodiment examines the effect of different temperatures during the activation step on the test. The specific experimental group design is shown in Table 6 below:

[0320] Table 6

[0321]

[0322] Experimental Procedure: The following experiments were conducted according to the one-step CUT&RUNLT experimental procedure of this invention. For detailed operation, please refer to the CUT&RUNLT procedure description of this invention in the experimental method above. The following is a brief description of the steps:

[0323] 1. Cell preparation

[0324] 1-1) Take 700,000 freshly harvested 293F cells, divide them into 7 equal parts, place them 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;

[0325] 1-2) Add 1 mL of washing buffer to the cell pellet from step 1-1), gently pipette to mix, centrifuge at 600 g for 5 min at room temperature, carefully discard the supernatant to obtain the cell pellet;

[0326] 1-3) Add 90 μL of washing buffer to the cell pellet from step 1-2) to resuspend the cells.

[0327] 2. Preparation and cell binding of ConA magnetic beads

[0328] 3. ConA magnetic beads - cell binding to primary antibody

[0329] 1) Dilute the Pol-II primary antibody with antibody buffer. Each experimental sample requires 50 μL of primary antibody dilution solution. Keep on ice for later use.

[0330] 2) Place the centrifuge tube containing the ConA magnetic bead-cell mixture from step 2 on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely separated from the liquid, carefully discard the supernatant, remove the centrifuge tube, add 50 μL of Pol-II primary antibody dilution solution, and incubate at room temperature for 2 hours by rotation.

[0331] 4. ConA magnetic beads - cell washing

[0332] 5. Binding and washing of pAG-MNase

[0333] 6. Fragmentation reaction and chromatin release

[0334] 1) Groups 1 to 7 in Table 6 above all used an activation system including: 50 μL washing buffer, DMSO with a final concentration of 10%, and CaCl2 with a final concentration of 1mM.

[0335] 2) After adding the activation system and gently mixing the cells, incubate the following groups simultaneously for 60 min: Group 1 at 0℃ (on ice), Group 2 at 4℃, Group 3 at 10℃, Group 4 at 15℃, Group 5 at 20℃, Group 6 at 25℃, and Group 7 at 30℃. This process allows for the release of nucleic acids while fragmenting the cells.

[0336] 3) Place the centrifuge tube from the previous step on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely separated from the liquid, carefully transfer the supernatant to a new PCR tube to obtain the fragmented nucleic acid.

[0337] 7. DNA extraction

[0338] 8. Library Construction

[0339] The same number of cycles was used for library amplification, with groups 1 to 7 above amplifying for 14 cycles each.

[0340] 9. Library sequencing

[0341] 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 6 As shown in Table 7, the peak number results for different activation temperature groups in the 10% DMSO activation system are as follows:

[0342] Table 7

[0343]

[0344] The results showed that in the experimental group (pol-II antibody) with a final concentration of 10% DMSO, the library yield increased with increasing activation temperature within the range of 0℃ to 30℃. Specifically, the number of peaks increased between 4℃ and 20℃, while it decreased between 20℃ and 30℃ (as shown in Table 7). From the IGV peak diagram ( Figure 6 As you can see, at 0℃, the effect is slightly worse, the nucleic acid fragments cannot be fully released, the signal is weaker, and the background is relatively high; at 4℃~30℃, the peak shape results with higher signal-to-noise ratio can be obtained for all groups.

[0345] Example 4: Comparison of the one-step and two-step activation processes using CUT&RUNLT with the traditional CUT&RUN method.

[0346] Example 4 compares the results of the CUT&RUNLT one-step activation and two-step activation processes with the traditional CUT&RUN method. The specific experimental group design is shown in Table 8 below:

[0347] Table 8

[0348]

[0349] Experimental Procedure: Unlike the above embodiments, this embodiment's experiments are conducted using the traditional CUT&RUN process, the one-step CUT&RUNLT method of this invention, and the two-step CUT&RUNLT method. For detailed instructions, please refer to the descriptions in the above experimental methods. The following is a brief summary of the steps:

[0350] In Table 8 above, Groups 1 and 2 are implemented using the traditional CUT&RUN method, Groups 3 and 4 are implemented using the one-step CUT&RUNLT method, and Groups 5 and 6 are implemented using the two-step CUT&RUNLT method. The specific operations are as follows:

[0351] 1. Cell preparation

[0352] 1-1) Take 600,000 freshly harvested 293F cells, divide them into 6 equal parts, place them in 1.5 mL centrifuge tubes, 100,000 cells / sample, centrifuge at 600 g for 5 min at room temperature, carefully discard the supernatant, and obtain the cell pellet;

[0353] 1-2) Add 1 mL of washing buffer to the cell pellet from step 1-1), gently pipette to mix, centrifuge at 600 g for 5 min at room temperature, carefully discard the supernatant to obtain the cell pellet;

[0354] 1-3) Add 90 μL of washing buffer to the cell pellet from step 1-2) to resuspend the cells.

[0355] The above 6 cell suspensions were designated as groups 1 to 6, and groups 1 and 2 were subjected to the traditional CUT & RUN method.

[0356] 2. Preparation of ConA magnetic beads and binding with cells to obtain a ConA magnetic bead-cell mixture;

[0357] 3. ConA magnetic beads - cell and primary antibody binding:

[0358] 1) In Group 1 of Table 8 above, the Pol-II primary antibody was diluted with antibody buffer. Each experimental sample required 50 μL of primary antibody dilution solution and should be kept on ice for later use. In Group 2, the CTCF primary antibody was diluted with antibody buffer. Each experimental sample required 50 μL of primary antibody dilution solution and should be kept on ice for later use.

[0359] 2) Place the centrifuge tube containing the ConA magnetic bead-cell mixture from step 2 on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely separated from the liquid, carefully discard the supernatant, remove the centrifuge tube, and add 50 μL of the corresponding Pol-II or CTCF primary antibody dilution solution to group 1 and group 2 respectively. Incubate at room temperature for 2 hours by rotation.

[0360] 4. ConA magnetic beads - cell washing;

[0361] 5. Binding and washing of pAG-MNase;

[0362] 6. Fragmentation reaction;

[0363] 7. Release of chromatin fragments;

[0364] 8. DNA extraction;

[0365] 9. Library Construction:

[0366] The same number of cycles was used for library amplification, with 14 cycles for each library.

[0367] 10. Library sequencing.

[0368] For groups 3 and 4 in Table 8 above, the CUT&RUNLT one-step method is adopted. The steps 1-5 involved are basically the same as those in this embodiment. The difference is in the process after step 5, which includes the following steps:

[0369] 6. Fragmentation reaction and chromatin release (one-step method):

[0370] 1) The activation system used in both Group 3 and Group 4 included 50 μL of washing buffer, 10% DMSO and 1mM CaCl2.

[0371] 2) After adding the activation system and gently mixing the cells, place them on a PCR instrument at 18°C ​​and incubate for 60 min to release nucleic acids while fragmenting.

[0372] 3) 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.

[0373] 7. DNA extraction;

[0374] 8. Library Construction:

[0375] The same number of cycles was used for library amplification, with 14 cycles for each library.

[0376] 9. Library sequencing.

[0377] For groups 5 and 6 in Table 8 above, the CUT & RUNLT two-step method is adopted. The steps 1-5 involved are basically the same as those in this embodiment. The difference is in the process after step 5, which includes the following steps:

[0378] 6. Fragmentation reaction and chromatin release (two-step method)

[0379] 1) Both Group 5 and Group 6 used an activation system consisting of 50 μL of washing buffer, 10% DMSO, and 1mM CaCl2.

[0380] 2) After adding the activation system and gently mixing the cells, place them on a PCR instrument at 4°C and incubate for 40 min.

[0381] 3) After incubation, add 10 mM EDTA and 2 mM EGTA to the reaction system, incubate at 18°C ​​for 50 min, terminate the reaction and release nucleic acids;

[0382] 4) Place the centrifuge tube from the previous step on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely separated from the liquid, carefully transfer the supernatant to a new PCR tube to obtain the fragmented nucleic acid.

[0383] 7. DNA extraction;

[0384] 8. Library Construction:

[0385] The same number of cycles was used for library amplification, with 14 cycles for each library.

[0386] 9. Library sequencing:

[0387] 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 7-8 As shown in Table 9, the number of peaks determined by different cut & run methods is as follows:

[0388] Table 9

[0389]

[0390] The results show that, based on the peak counts in Table 9, the peak counts in both CUT&RUNLT methods (one-step CUT&RUNLT and two-step CUT&RUNLT) are significantly higher than those in the traditional CUT&RUN experiment. From the IGV peak diagrams, in the antibody Pol-II and antibody CTCF groups, compared with the traditional CUT&RUN, the signal-to-noise ratio of the one-step or two-step CUT&RUNLT method of this invention is significantly improved, and the peaks are more complete. Figures 7-8 It can be seen that under low-temperature activation and the addition of a release promoter (such as DMSO), both CUT&RUNLT implementation schemes (one-step method with simultaneous activation and release, and two-step method with separate activation and release) can achieve good results, which are significantly better than the results of traditional CUT&RUN experiments.

[0391] Example 5: Effects of pAG-Benzonase nuclease on different activation systems

[0392] This embodiment investigates the effect of pAG-Benzonase nuclease in activation systems with different DMF concentrations and compares its effects with pAG-MNase and pAG-DNase I. The specific experimental group design is shown in Table 10 below:

[0393] Table 10

[0394]

[0395] Experimental Procedure: The following experiment follows the CUT&RUNLT one-step experimental procedure. For detailed instructions, please refer to the description in the Experimental Methods section above. The following is a brief summary of the steps:

[0396] 1. Cell preparation:

[0397] 1) Take 2.2 million freshly harvested 293F cells, divide them into 22 equal parts, place them 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;

[0398] 2) Add 1 mL of washing buffer to the cell pellet from the previous step, gently pipette to mix, centrifuge at 600g for 5 min at room temperature, and carefully discard the supernatant;

[0399] 3) Resuspend the cells in the cell pellet from the previous step by adding 90 μL of washing buffer;

[0400] 2. Preparation and cell binding of ConA magnetic beads;

[0401] 3. ConA magnetic beads - cell and primary antibody binding:

[0402] 1) In the groups 1-11 of Table 10 above, the Pol-II primary antibody was diluted with antibody buffer. Each experimental sample required 50 μL of primary antibody dilution solution and should be kept on ice for later use. In the groups 12-22, the CTCF primary antibody was diluted with antibody buffer. Each experimental sample required 50 μL of primary antibody dilution solution and should be kept on ice for later use.

[0403] 2) Place the centrifuge tube containing the ConA magnetic bead-cell mixture from step 2 on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely separated from the liquid, carefully discard the supernatant, remove the centrifuge tube, and add 50 μL of the corresponding Pol-II or CTCF primary antibody dilution solution to groups 1-11 and 12-22 respectively. Incubate at room temperature for 2 hours by rotation.

[0404] 4. ConA magnetic beads - cell washing;

[0405] 5. Binding and washing of pAG-Benzonase, pAG-MNase, and pAG-DNase I:

[0406] In Table 10 above, groups 1-9 and 12-20 used pAG-Benzonase as the nuclease, groups 10 and 21 used pAG-MNase as the nuclease, and groups 11 and 22 used pAG-DNase I as the nuclease.

[0407] 5-1) Remove the centrifuge tubes from the magnetic rack. Add 50 μL of Dig-wash buffer and pAG-Benzonase to groups 1-9 and 12-20 to a final concentration of 200 ng / mL. Add 50 μL of Dig-wash buffer and pAG-MNase to groups 10 and 21 to a final concentration of 200 ng / mL. Add 50 μL of Dig-wash buffer and pAG-DNase I to groups 11 and 22 to groups 20. After gently resuspending the ConA magnetic bead-cell mixture by pipetting, incubate at 4°C for 1 h.

[0408] 5-2) Place the centrifuge tubes incubated with pAG-Benzonase, pAG-MNase, and pAG-DNase I respectively on a magnetic rack and let them stand for 2 minutes. After the magnetic beads are completely separated from the liquid, carefully discard the supernatant.

[0409] 5-3) Remove the centrifuge tube from the magnetic rack, add 200 μL of Dig-washing buffer, resuspend the ConA magnetic bead-cell mixture, gently pipette to mix, and let stand at room temperature for 2 min;

[0410] 5-4) Place it on a magnetic rack and let it stand for 2 minutes until the magnetic beads are completely separated from the liquid. Then carefully discard the supernatant.

[0411] 5-5) Repeat steps 5-3) and 5-4) once.

[0412] 6. Fragmentation reaction and chromatin release

[0413] 1) Groups 1 and 12: Activation system consists of 50 μL wash buffer, final concentration of 1 mM MgCl2, without added DMF; Groups 2 and 13: Activation system consists of 50 μL wash buffer, final concentration of 0.3% DMF and final concentration of 1 mM MgCl2; Groups 3 and 14: Activation system consists of 50 μL wash buffer, final concentration of 2.5% DMF and final concentration of 1 mM MgCl2; Groups 4 and 15: Activation system consists of 50 μL wash buffer, final concentration of 5% DMF and final concentration of 1 mM MgCl2; Groups 5 and 16: Activation system consists of 50 μL wash buffer, final concentration of 10% DMF and final concentration of 1 mM MgCl2; Groups 6 and 17: Activation system consists of 50 μL wash buffer, final concentration of 15% DMF and final concentration of 1 mM MgCl2; Groups 7 and 18: Activation system consists of 50 μL wash buffer... The activation system for groups 8 and 19 consisted of 50 μL of wash buffer, 25% DMF, and 1 mM MgCl2; for groups 9 and 20, the activation system consisted of 50 μL of wash buffer, 30% DMF, and 1 mM MgCl2; for groups 10 and 21, the activation system consisted of 50 μL of wash buffer, 15% DMF, and 1 mM CaCl2; and for groups 11 and 22, the activation system consisted of 50 μL of wash buffer, 15% DMF, and 1 mM MgCl2.

[0414] 2) After gently mixing the cells by pipetting, all groups were incubated at 18°C ​​for 60 min on a PCR instrument to release nucleic acids during fragmentation;

[0415] 3) 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.

[0416] 7. DNA extraction;

[0417] 8. Library Construction:

[0418] The same number of cycles was used for library amplification, with 14 cycles for each library.

[0419] 9. Library sequencing:

[0420] The purified library products were sent to a sequencing company for sequencing. Sequencing results were analyzed on the Galaxy website (https: / / usegalaxy.org / ), and target protein binding maps were viewed using IGV software. The sequencing results are shown in Table 11. Figure 9 A- Figure 9 As shown in B.

[0421] Table 11

[0422]

[0423] The results showed that, in experiments with both Pol-II and CTCF antibodies, at the same sequencing volume (1G), the number of peaks in both antibody groups significantly increased after adding DMF at final concentrations of 0.3%–30% compared to those without DMF (Table 11). Furthermore, in the IGV peak diagram, the addition of DMF enhanced the peak signals, reduced the background, and improved the signal-to-noise ratio in both antibody groups. Figure 9 A- Figure 9 B). The results show that the release of nucleic acid fragments can be enhanced within the final DMF concentration range of 0.3% to 30%.

[0424] In the activation system with 15% DMF, the peak numbers of the three nucleases pAG-Benzonase, pAG-MNase, and pAG-DNase I were similar. The IGV peak diagram showed that the peak shape and signal-to-noise ratio of the three nucleases were generally similar.

[0425] Example 6: The effect of DMSO addition on the results of CUT & RUN experiments on cell nuclear samples

[0426] This embodiment examines the effect of DMSO addition on the results of CUT & RUN experiments on cell nuclear samples. The specific experimental group design is shown in Table 12:

[0427] Table 12

[0428]

[0429] Experimental Procedure: The following experiment follows the CUT&RUNLT one-step experimental procedure. For detailed instructions, please refer to the description in the Experimental Methods section above. The following is a brief summary of the steps:

[0430] Preparation of cell nucleus

[0431] Take 800,000 freshly harvested 293F cells, divide them into 8 equal parts, place them in 1.5 mL centrifuge tubes, 100,000 cells / sample, centrifuge at 600 g for 5 min at room temperature, and carefully discard the culture medium supernatant;

[0432] Add 1 mL of lysis buffer (formulation in Experimental Methods) to the cell pellet, gently pipette to mix, and incubate on ice for 5 min to lyse the cells.

[0433] Centrifuge at 600 g for 5 min at 4℃, carefully discard the supernatant, and obtain the cell nucleus precipitate;

[0434] Add 1 mL of washing buffer to the nuclear pellet from the previous step, gently pipette to mix, centrifuge at 600g for 5 min at room temperature, carefully discard the supernatant to obtain the nuclear pellet;

[0435] Add 90 μL of washing buffer to the cell nucleus pellet from the previous step to resuspend the cell nuclei.

[0436] Preparation of ConA magnetic beads and binding to the cell nucleus

[0437] ConA magnetic beads - cell nucleus and primary antibody binding

[0438] ConA magnetic beads - washing of cell nuclei

[0439] binding and washing of pAG-MNase

[0440] Fragmentation reaction and chromatin release

[0441] 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.

[0442] 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.

[0443] DNA extraction

[0444] Library Construction

[0445] Library amplification used the same number of cycles, with groups 1-8 all undergoing 14 cycles of amplification.

[0446] Library sequencing

[0447] 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:

[0448] Table 13

[0449]

[0450] 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.

[0451] Example 7: Effect of different protein-nucleic acid fragment release agents on CUT&RUN experimental results

[0452] This example investigates the effects of adding different proteolytic nucleic acid fragment release agents, such as Tween-20, NP40 (or IGEPA CA-630), Triton X-100, CHAPS, and sodium deoxycholate, on the results of the CUT&RUN experiment. The specific experimental group design is shown in Table 14.

[0453] Table 14

[0454]

[0455] Experimental Procedure: The following experiment follows the CUT&RUNLT one-step experimental procedure. Please refer to the description above for detailed operation. The following is a brief summary of the procedure:

[0456] 1. Cell preparation

[0457] 1) Take 1 million freshly harvested 293F cells, divide them into 12 equal parts and place them in 1.5 mL centrifuge tubes. Centrifuge at 600 g for 5 min at room temperature, 100,000 cells / sample, and carefully discard the supernatant.

[0458] 2) Add 1 mL of washing buffer to the cell pellet from the previous step, gently pipette to mix, centrifuge at 600g for 5 min at room temperature, carefully discard the supernatant to obtain the cell pellet;

[0459] 3) Resuspend the cells in the cell pellet from the previous step by adding 90 μL of washing buffer;

[0460] 2. Preparation and cell binding of ConA magnetic beads;

[0461] 3. ConA magnetic beads - cell and primary antibody binding:

[0462] 1) Dilute the Pol-II primary antibody with antibody buffer. Each experimental sample requires 50 μL of primary antibody dilution solution. Keep on ice for later use.

[0463] 2) Place the centrifuge tube containing the ConA magnetic bead-cell mixture from step 2 on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely separated from the liquid, carefully discard the supernatant, remove the centrifuge tube, add 50 μL of Pol-II primary antibody dilution buffer, and incubate at room temperature for 2 hours by rotation.

[0464] 4. ConA magnetic beads - cell washing;

[0465] 5. Binding and washing of pAG-MNase;

[0466] 6. Fragmentation reaction and chromatin release:

[0467] 1) The activation systems for groups 1-6 in Table 14 above consisted of 50 μL of wash buffer, a final concentration of 0.05% of digitalis saponin, Tween-20, NP40, Triton X-100, CHAPS, or sodium deoxycholate, and a final concentration of 1 mM CaCl2, respectively. The activation systems for groups 7-12 consisted of 50 μL of wash buffer, a final concentration of 0.5% of digitalis saponin, Tween-20, NP40, Triton X-100, CHAPS, or sodium deoxycholate, and a final concentration of 1 mM CaCl2. 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.

[0468] 2) 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.

[0469] 7. DNA extraction;

[0470] 8. Library Construction:

[0471] The same number of cycles was used for library amplification, with groups 1 to 12 amplifying for 14 cycles each;

[0472] 9. Library sequencing:

[0473] The purified library products were sent to a sequencing company for sequencing. Sequencing results were analyzed on the Galaxy website (https: / / usegalaxy.org / ). Target protein binding maps were viewed using IGV software. Sequencing result analysis is as follows: Figure 11 As shown in Table 15, the number of library peaks after adding different detergents is as follows:

[0474] Table 15

[0475]

[0476] The results showed that, based on the peak counts in Table 15, the number of peaks in the group with the addition of 0.05% of the protein-nucleic acid fragment complex release agent was slightly lower than that in the group with the addition of 0.5%. From the IGV peak diagram, high signal-to-noise ratio CUT&RUN results could be obtained after adding different protein-nucleic acid fragment complex release agents. Figure 11 Therefore, the aforementioned common protein-nucleic acid fragment release agents can all achieve the effect of promoting the release of protein-nucleic acid fragment complexes.

[0477] Example 8: Effects of different release promoters on CUT&RUN experimental results

[0478] This example investigates the effects of adding different release promoters, streptococcal hemolysin O and melittin, on the results of the CUT&RUN experiment. The specific experimental group design is shown in Table 16.

[0479] Table 16

[0480]

[0481] The experimental process in this embodiment is basically the same as that in embodiment 7, except for step 6, which is shown in the following details:

[0482] 6. Fragmentation reaction and chromatin release

[0483] 1) In Table 16 above, the activation system for Group 1 consists of 50 μL wash buffer, 5% DMSO, and 1mM CaCl2; the activation system for Group 2 consists of 50 μL wash buffer, 10 ng / mL Streptococcal Hemolysin O (SLO), and 1mM CaCl2; the activation system for Group 3 consists of 50 μL wash buffer, 50 ng / mL SLO, and 1mM CaCl2; the activation system for Group 4 consists of 50 μL wash buffer, 100 ng / mL SLO, and 1mM CaCl2; the activation system for Group 5 consists of 50 μL wash buffer, 0.01 μM melittin, and 1mM CaCl2; the activation system for Group 6 consists of 50 μL wash buffer, 0.1 μM melittin, and 1mM CaCl2; and the activation system for Group 7 consists of 50 μL wash buffer... Washing buffer, melitoxin at a final concentration of 1 μM, and CaCl2 at a final concentration of 1 mM. After gently mixing the cells by pipetting, incubate at 18°C ​​for 60 min on a PCR instrument to release nucleic acids during fragmentation.

[0484] 2) Place the centrifuge tube from the previous step on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely separated from the liquid, carefully transfer the supernatant to a new PCR tube to obtain the fragmented nucleic acid.

[0485] Subsequent experimental procedures were the same as in Example 7. Sequencing results analysis is as follows: Figure 12 As shown in Table 17, the number of peaks in the library after the addition of different release promoters is as follows:

[0486] Table 17

[0487]

[0488] The results showed that, based on the number of peaks in Table 17, there was no significant difference in the number of peaks between the groups with the addition of 5% DMSO and those with the addition of streptococcal hemolysin O or melittin. Furthermore, at all three different concentrations, the peak signals were similar and the signal-to-noise ratios were comparable after adding streptococcal hemolysin O or melittin in the IGV peak diagram. Figure 12 Therefore, adding different concentrations of release promoters during the activation step: both streptococcal hemolysin O and melitoxin can promote the release of targeted cleaved nucleic acid fragments into the extracellular supernatant.

[0489] Example 9: Effect of different release promoter combinations on CUT&RUN experimental results

[0490] This embodiment examines the effect of adding different combinations of release promoters on the results of the CUT&RUN experiment. The specific experimental group design is shown in Table 18:

[0491] Table 18

[0492]

[0493] The experimental process in this embodiment is basically the same as that in embodiment 8, except for step 6, which is shown in the following details:

[0494] 6. Fragmentation reaction and chromatin release

[0495] 1) In Table 18 above, the activation system for Group 1 consists of 50 μL wash buffer, 5% DMSO, and 1 mM CaCl2; the activation system for Group 2 consists of 50 μL wash buffer, 5% DMSO, 0.05% digitalis saponin, and 1 mM CaCl2; the activation system for Group 3 consists of 50 μL wash buffer, 5% DMSO, 0.05% Tween-20, and 1 mM CaCl2; the activation system for Group 4 consists of 50 μL wash buffer, 5% DMSO, 0.05% NP40, and 1 mM CaCl2; the activation system for Group 5 consists of 50 μL wash buffer, 5% DMSO, 0.05% Triton X-100, and 1 mM CaCl2; and the activation system for Group 6 consists of 50 μL wash buffer... Washing buffer, final concentration of 5% DMSO and final concentration of 0.05% CHAPS, and final concentration of 1mM CaCl2; Group 7 activation system consists of 50 μL washing buffer, final concentration of 5% DMSO and final concentration of 0.05% sodium deoxycholate, and final concentration of 1mM CaCl2. Group 8 activation system consisted of 50 μL wash buffer, 0.05% final concentration of NP40 and 0.05% final concentration of digitalis saponins, and 1 mM CaCl2; Group 9 activation system consisted of 50 μL wash buffer, 0.05% final concentration of NP40 and 0.05% final concentration of Tween-20; Group 10 activation system consisted of 50 μL wash buffer, 0.05% final concentration of NP40 and 0.05% final concentration of Triton X-100; Group 11 activation system consisted of 50 μL wash buffer, 0.05% final concentration of NP40 and 0.05% final concentration of CHAPS; Group 12 activation system consisted of 50 μL wash buffer, 0.05% final concentration of NP40 and 0.05% final concentration of sodium deoxycholate.

[0496] After gently mixing the cells by pipetting, incubate them at 18°C ​​for 60 min on a PCR instrument to release nucleic acids during fragmentation.

[0497] 2) Place the centrifuge tube from the previous step on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely separated from the liquid, carefully transfer the supernatant to a new PCR tube to obtain the fragmented nucleic acid.

[0498] Subsequent experimental procedures were the same as in Example 8. Sequencing results analysis is as follows: Figure 13 As shown in Table 19, the number of peaks in the library after adding different combinations of release promoters is as follows:

[0499] Table 19

[0500]

[0501] The results show that, based on the number of peaks in Table 19, there is no significant difference in the number of peaks between the groups with 5% DMSO added alone and the groups with the combination of two release promoters. The peak signals are similar in the IGV peak diagram, and the signal-to-noise ratios are also similar. Figure 13 Therefore, adding different release promoters in the activation step can promote the release of targeted cleaved nucleic acid fragments into the extracellular supernatant.

[0502] Example 10: Effects of different temperature gradients on the addition of release promoters after activation on cut & run experiment results

[0503] This embodiment examines the effects of different temperature gradients on activation and the addition of release promoters after activation on the results of CUT&RUN experiments. The specific experimental group design is shown in Table 20.

[0504] Table 20

[0505]

[0506] Experimental Procedure: The following experiment follows the CUT&RUNLT one-step experimental procedure. For detailed instructions, please refer to the description in the Experimental Methods section above. The following is a brief summary of the steps:

[0507] 1. Cell preparation:

[0508] 1) Take 600,000 freshly harvested 293F cells, divide them into 6 equal parts and place them in 1.5 mL centrifuge tubes. Centrifuge at 600 g for 5 min at room temperature, 100,000 cells / sample, and carefully discard the supernatant.

[0509] 2) Add 1 mL of washing buffer to the cell pellet from the previous step, gently pipette to mix, centrifuge at 600g for 5 min at room temperature, and carefully discard the supernatant;

[0510] 3) Resuspend the cells in the cell pellet from the previous step by adding 90 μL of washing buffer;

[0511] 2. Preparation and cell binding of ConA magnetic beads;

[0512] 3. ConA magnetic beads - cell and primary antibody binding:

[0513] 1) In Groups 1, 3, and 5 of Table 20 above, Pol-II primary antibody was diluted with antibody buffer. Each experimental sample required 50 μL of primary antibody dilution solution and should be kept on ice for later use. In Groups 2, 4, and 6, CTCF primary antibody was diluted with antibody buffer. Each experimental sample required 50 μL of primary antibody dilution solution and should be kept on ice for later use.

[0514] 2) Place the centrifuge tube containing the ConA magnetic bead-cell mixture from step 2 on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely separated from the liquid, carefully discard the supernatant, remove the centrifuge tube, and add 50 μL of the corresponding Pol-II or CTCF primary antibody dilution solution according to the group number. Incubate at room temperature for 2 hours by rotation.

[0515] 4. ConA magnetic beads - cell washing;

[0516] 5. Binding and washing of pAG-MNase;

[0517] 6. Fragmentation reaction and chromatin release;

[0518] 1) The activation system for groups 1-4 consisted of 50 μL of wash buffer, 1 mM CaCl2, and 5% DMSO; the activation system for groups 5 and 6 consisted of 50 μL of wash buffer and 1 mM CaCl2, without any release promoter.

[0519] 2) After gently mixing the cells by pipetting, all groups were fragmented and released on a PCR instrument according to the following procedure:

[0520] Group 1 and Group 2: Incubate at 0℃ for 20 min, at 4℃ for 20 min, and at 10℃ for 20 min.

[0521] Groups 3 and 4: Activation temperature 18℃, incubation for 60 min, nucleic acid release during fragmentation;

[0522] Groups 5 and 6: Activation temperature 18℃, incubation for 40 min, add 5% DMSO to the system, incubate at 18℃ for 20 min to release nucleic acid fragments;

[0523] 3) 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.

[0524] 7. DNA extraction;

[0525] 8. Library Construction:

[0526] The same number of cycles was used for library amplification, with 14 cycles for each library.

[0527] 9. Library sequencing:

[0528] The purified library products were sent to a sequencing company for sequencing. Sequencing results were analyzed on the Galaxy website (https: / / usegalaxy.org / ). Target protein binding maps were viewed using IGV software. Sequencing result analysis is as follows: Figure 14 As shown in Table 21, the number of peaks in different library groups is as follows:

[0529] Table 21

[0530]

[0531] 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.

[0532] 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 for constructing a nucleic acid library based on low-temperature release of nucleic acid fragments in a CUT&RUN process, characterized in that, It includes the following steps: S1. Provide a cell or cell nucleus sample; S2. Wash or resuspend the cell or cell nucleus sample to obtain a cell or cell nucleus suspension; S3. Pretreat the cell or cell nucleus suspension to obtain a cell or cell nucleus precipitate; S4. Treat the cell or cell nucleus precipitate with an antibody to obtain an antibody-cell or antibody-cell nucleus complex; S5. Treat the antibody-cell or antibody-cell nucleus complex with a nuclease to obtain a nuclease-antibody-cell or nuclease-antibody-cell nucleus complex; S6. Under the temperature T for releasing nucleic acid fragments at low temperature, in an activation release reaction system containing a metal ion buffer solution, use a protein nucleic acid fragment complex releasing agent to perform fragmentation treatment of nucleic acids and release nucleic acid fragments at low temperature on the nuclease-antibody-cell or nuclease-antibody-cell nucleus complex; wherein, the temperature T is a low temperature of 10-25 °C; the treatment time t is 20-240 min; S7. Extract the released nucleic acid fragments and prepare a nucleic acid library; Among them, in S7, the extraction includes: separating and taking the supernatant of the nucleic acid fragments released in S6, and adding a DNA extraction reagent to the supernatant for DNA extraction; Among them, the protein nucleic acid fragment complex releasing agent is selected from the following group: digitonin, DMSO, DMF, Tween-20, NP40, TritonX-100, CHAPS, sodium deoxycholate, streptolysin O, melittin, or a combination thereof; The concentration of the protein nucleic acid fragment complex releasing agent is 0.01% - 50% (v / v); The concentration of the metal ions in the metal ion buffer solution is 0.01 mM - 10 mM; The nuclease is fused with a domain or polypeptide capable of binding an antibody, and the nuclease is targeted and fixed near the target protein; the domain or polypeptide capable of binding an antibody is selected from the following group: protein A, protein G, protein L, or a combination thereof; The activation release reaction system includes: a protein nucleic acid fragment complex releasing agent and a metal ion buffer solution with a concentration of 0.01 mM - 10 mM; The DNA extraction reagent includes EDTA, SDS, proteinase K; The nuclease is selected from DNase I, MNase, Benzonase.

2. The method as described in claim 1, characterized in that, The nuclease is selected from: pAG-DNase I, pAG-MNase, pAG-Benzonase.

3. The method as described in claim 1, characterized in that, In the process of the fragmentation treatment of nucleic acids in step S6, the temperature T 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 continuously reacting at different temperatures respectively.

4. The method as described in claim 1, characterized in that, The metal ions in the metal ion buffer solution are selected from the following group: calcium ions, magnesium ions, manganese ions, iron ions, zinc ions, cobalt ions, or a combination thereof.

5. The method as described in claim 1, characterized in that, The DNA extraction reagent includes: EDTA, SDS and proteinase K.

6. The method as described in claim 1, characterized in that, The method constructs a nucleic acid library using the following kit, and the kit includes: (1) A protein nucleic acid fragment complex releasing agent; (2) A metal ion buffer solution; (3) Cell washing buffer; and (4) Fragmentation reaction reagents; the fragmentation reaction reagents include nucleases.

7. A method for measuring protein-nucleic acid interactions, characterized in that, include: (S1) Construct a nucleic acid library using the method described in claim 1; and (S2) Sequencing of the nucleic acid library to generate multiple sequencing reads for measuring protein-nucleic acid interactions.

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