A ChIP-seq chromatin immunoprecipitation method for important economic organs of plants

Abstract

The present invention relates to a ChIP-seq chromatin immunoprecipitation method for plant economic organs, and provides a new aChIP experimental technology to solve the problem that existing methods are difficult to produce qualified DNA-protein interaction data in materials such as seeds, fruits and tubers. On the basis of ensuring the efficiency of chromatin separation, centrifugal precipitation and improved buffer formula are used to remove complex cellular inclusions in the material, and the chromatin is concentrated to efficiently obtain clean, high-concentration fragmented chromatin, and finally obtain high-quality ChIP-seq data in plant economic organs. This method is fast, efficient, sensitive, high-quality, and widely applicable. It is not only suitable for conventional plant tissues, but also for plant economic organs rich in substances such as oils, starch, sugars, organic acids and phenols. It expands the research on epigenetics, functional genomics and gene transcription regulation in the fields of plant seed development, dormancy and germination, fruit ripening and nutrient accumulation.

Description

Technical Field

[0001] The invention relates to the field of molecular biology, and in particular to a ChIP-seq chromatin immunoprecipitation method for important economic organs of plants. Background Art

[0002] As more and more plant genomes are sequenced and assembled, completing the annotation of the corresponding functional genomes and systematically identifying cis-regulatory elements and their gene expression regulatory networks is one of the important tasks in the post-genomic era. Chromatin immunoprecipitation sequencing (ChIP-seq) is a common experimental method for studying the interaction between DNA and proteins in the cell nucleus. It is widely used in the study of transcription factor binding and histone modification sites, and is an important experimental method in the field of epigenetics and gene transcription regulation research.

[0003] The yield and quality of grain are strategic needs to ensure agricultural security and improve the national economy and people's livelihood. With the increase in people's demand for high-quality life, the study of grain crop yield and quality is becoming more and more important. At present, there are few reports on the epigenetics of economic organs of important crops (seeds, fruits, flowers, stems and tubers, etc.), as well as the genome-wide binding sites of important transcription factors related to yield and quality formation. According to the statistical results of published articles, only 3.5% of the existing ChIP-seq data involve plant economic organs, and the data quality is seriously low, and low-quality data will seriously affect the direction of data analysis and the credibility of the final conclusion. We believe that one of the important reasons is that the existing conventional ChIP-seq and CUT&Tag experimental methods in plants cannot effectively produce high-quality ChIP-seq data in economic organs rich in sugars, phenols, starch, oils and organic acids. This seriously limits the research on epigenetic and gene transcription regulation in the fields of plant seed development, dormancy and germination, fruit ripening and nutrient accumulation. Summary of the invention

[0004] The present invention proposes a ChIP-seq chromatin immunoprecipitation method for important economic organs of plants. In view of the problem that existing experimental methods are difficult to produce qualified DNA-protein interaction data in special materials such as seeds, fruits, tubers and flowers, a new aChIP experimental method that can be used for important economic organs of plants is provided, which can be simple, efficient and high-quality in seeds, fruits, tubers and flowers. High-quality data is produced in important economic organs of plants such as flowers, greatly expanding the research of epigenetics and gene transcription regulation in this field. The aChIP method can significantly improve the efficiency of chromatin immunoprecipitation by abandoning the steps of extracting cell nuclei in the conventional plant ChIP-seq method and cleaning the cell contents, thereby increasing the concentration and purity of fragmented chromatin entering the immunoprecipitation. At the same time, aChIP identifies more peaks in conventional materials (such as young leaves and seedlings), and produces high-quality ChIP-seq data such as chromatin enzymes DME or other transcription factors that bind weakly to DNA. It further provides important technical support for the field of epigenetic research. At the same time, it can lay a technical foundation for three-dimensional genomics experiments on important economic organs of plants, such as ChIA-PET, HiChIP, Hi-C, etc.

[0005] The present invention provides a ChIP-seq chromatin immunoprecipitation method, comprising the following steps: (1) collecting materials and performing formaldehyde cross-linking fixation; (2) sequentially adding washing buffer Z1, buffer Z2, buffer Z3 and buffer Z4, removing cell contents by low-speed centrifugation, and concentrating chromatin; (3) using ultrasound to fragment chromatin; and (4) using the fragmented chromatin for subsequent construction of a ChIP-seq library.

[0006] Furthermore, the step (1) is to chop the collected material into pieces, place it in a PBS solution, cross-link and fix it with formaldehyde under a vacuum environment, evacuate the gas 2-3 times and stir to mix well to ensure that the material is fully cross-linked and fixed, and place the material in a glycine solution to terminate the cross-linking.

[0007] Furthermore, in step (2), buffer Z1 comprises: 20-100 mM HEPES, 100-300 mM NaCl, 0.2-2 mM EDTA, 0.5-2% Triton X-100, 0.05-0.5% sodium deoxycholate, 0.1-0.5% SDS, 1x Protease Inhibitor.

[0008] Furthermore, in step (2), buffer Z2 comprises: 1xPBS, 50-200mM NaCl, 8-18.8% Hexylene glycol, 0.1-1% Triton X-100, 0.1-2% SDS, 0.01-0.2% 2-Mercaptoethanol, 0.1-2mM EDTA, 1-20mM KCl.

[0009] Furthermore, after adding buffer Z2, the centrifugal speed during cleaning is set at 200-800g according to the physical characteristics of different plant economic organs.

[0010] Furthermore, in step (2), buffer Z3 comprises: 20-100 mM HEPES, 100-300 mM NaCl, 0.1-2 mM EDTA, 0.5-2% Tween-20, and 0.5-2% Triton X-100.

[0011] Furthermore, in step (2), buffer Z4 comprises: 20-100 mM HEPES, 100-300 mM NaCl, 0.1-2 mM EDTA, 0.5-2% Triton X-100, 0.01-0.2% Sodium deoxycholate, 0.01-0.3% SDS, 1x protease inhibitor.

[0012] Furthermore, the step (3) is performed with two rounds of ultrasonic disruption, wherein after the first round of ultrasonic disruption, the solution is centrifuged at 10,000 g for 10 min at 4°C, and the supernatant is placed in a new centrifuge tube for a second round of ultrasonic disruption, wherein each round of ultrasonic disruption is performed at 30 son / 50 s off; 32 cycles are performed; the chromatin solution after ultrasonic disruption is centrifuged again, and the solution is centrifuged at 10,000 g for 10 min at 4°C, and the clarified middle layer liquid is transferred to a new centrifuge tube (and care is taken to avoid the upper layer of oil and fat-like substances and the lower layer of precipitate-like substances).

[0013] Furthermore, the step (4) includes sequentially incubating the fragmented chromatin with antibodies, immunoprecipitating, cleaning non-specific binding, decrosslinking DNA-protein, purifying DNA and / or high-throughput sequencing.

[0014] The present invention also provides a cleaning solution for constructing a ChIP-seq library, the cleaning solution comprising: buffer Z1, buffer Z2, buffer Z3 and / or buffer Z4, wherein:

[0015] The buffer Z1 includes: 20-100 mM HEPES, 100-300 mM NaCl, 0.2-2 mM EDTA, 0.5-2% Triton X-100, 0.05-0.5% sodium deoxycholate, 0.1-0.5% SDS, 1x protease inhibitor;

[0016] The buffer Z2 includes: 1xPBS, 50-200mM NaCl, 8-18.8% Hexylene glycol, 0.1-1% Triton X-100, 0.1-2% SDS, 0.01-0.2% 2-Mercaptoethanol, 0.1-2mM EDTA, 1-20mM KCl;

[0017] The buffer Z3 includes: 20-100 mM HEPES, 100-300 mM NaCl, 0.1-2 mM EDTA, 0.5-2% Tween-20, 0.5-2% Triton X-100;

[0018] The buffer Z4 includes: 20-100 mM HEPES, 100-300 mM NaCl, 0.1-2 mM EDTA, 0.5-2% Triton X-100, 0.01-0.2% Sodium deoxycholate, 0.01-0.3% SDS, 1x protease inhibitor.

[0019] The present invention also provides the use of the cleaning solution in the construction of a ChIP-seq library.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention aims to solve the problem that existing experimental methods are difficult to produce qualified DNA-protein interaction data in special materials such as seeds, fruits, tubers and flowers, and provides a new aChIP experimental method; compared with the published ChIP-seq data of some important economic organs of plants, or the published CUT&Tag data of conventional materials, the data signal-to-noise ratio is significantly improved, the number of identified peaks is significantly increased, and the peak intensity is also significantly enhanced;

[0022] (2) The present invention abandons the step of extracting cell nuclei in the conventional plant ChIP-seq method and adds 4 different washing steps. On this basis, the efficiency of chromatin acquisition of plant economic organs is significantly improved. It is simple, efficient, and widely applicable. High-quality data can be produced in the 10 different important economic organs of plants that have been tested;

[0023] (3) The present invention efficiently obtains high-concentration and high-purity fragmented chromatin to enter the immunoprecipitation step, significantly improving the efficiency of immunoprecipitation, and the yield of ChIP-DNA is increased by 3-10 times in different materials;

[0024] (4) Due to the improvement of ChIP-DNA yield, the present invention can significantly increase the proportion of DNA in the target protein binding region in the total ChIP-DNA. Therefore, in conventional materials such as young leaves and seedlings, the present invention can identify more low-enrichment peaks.

[0025] (5) The present invention can significantly improve the data quality in ChIP-seq studies of plant transcription factors, DNA binding proteins, RNAPII and chromatin enzymes that are weakly bound to DNA, making the application scope of the present invention wider. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0027] Figure 1 It is a schematic diagram of the novel aChIP experimental method of the present invention.

[0028] Figure 2 This is a demonstration that the new aChIP experimental method of the present invention produces data in Brassica napus seeds that is superior to other methods.

[0029] Figure 3 This is a screenshot of the partial location of the genome in which the new aChIP experimental method of the present invention produces high-quality data in 9 different important economic organs of plants.

[0030] Figure 4 This is a screenshot of the partial location of the genome in which the new aChIP experimental method of the present invention is superior to the published data in potato tubers and tomato fruits.

[0031] Figure 5This is a screenshot of the partial location of the genome where the novel aChIP assay method of the present invention produces high-quality data in transcription factors, DNA binding proteins, RNAPII and chromatin enzymes.

[0032] Figure 6 This is a screenshot of the partial location of the genome where the new aChIP experimental method of the present invention identified more low-enrichment peaks in conventional materials.

[0033] Figure 7 This is a screenshot of the partial location of the genome where the new aChIP experimental method of the present invention is superior to the published CUT&Tag in conventional materials. DETAILED DESCRIPTION

[0034] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all belong to the scope of the present invention.

[0035] Example 1

[0036] The present invention proposes a ChIP-seq chromatin immunoprecipitation method suitable for tissues such as seeds, fruits, stems and tubers, aiming at the particularity of materials rich in oil, starch, protein, organic acid, sugar and phenol in important economic organs of plants. The method comprises the following steps:

[0037] 1. Collect important economic organs of different plants and fix them with formaldehyde for cross-linking

[0038] 1.1 Collect important economic organs of different plants, such as mature rapeseed and soybean seeds rich in oil and protein, rice and corn seeds with high starch content, carnation flowers rich in phenolic substances, sugarcane stalks with high sugar content, orange peel and pulp rich in sugar phenols and organic acids, etc. Chop the collected materials with a blade and place them in 1x PBS solution. Cross-link and fix with 1% formaldehyde in a vacuum environment for 10-15 minutes, vent gas 2-3 times and stir to ensure sufficient cross-linking and fixation of the material. Place the material in a 0.2M glycine solution to terminate cross-linking. Store the fixed material at -80℃ for long-term storage.

[0039] 1.2 Use liquid nitrogen and a mortar to fully grind the fixed plant important economic organ materials into powder. Note: Due to the particularity of its inherent physical form, plant important economic organs are difficult to grind and break. You can first smash the material into fine particles and then grind it several times. Make sure there are no large particles of material to avoid the continued release of complex cell contents in subsequent experimental steps.

[0040] 2. Add washing buffer in sequence and use low-speed centrifugation instead of nylon membrane filtration to remove the complex cellular contents in the important economic organs of plants, while ensuring the efficiency of chromatin separation and chromatin concentration.

[0041] 2.1 Take about 0.5-1g of the ground material, add 15mL of washing buffer Z1 (50mM HEPES, 150mM NaCl, 1mM EDTA, 1.25% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1x protease inhibitor), and rotate at 4℃, 10rpm for 10min. Next, use a large refrigerated centrifuge, 4℃, 500g non-fixed angle centrifuge for 5min, and carefully discard the supernatant to preliminarily remove the water-soluble contents in the plant economic organs.

[0042] 2.2 Take 15mL of washing buffer Z2 (1xPBS, 100mM NaCl, 12.8% Hexylene glycol, 0.5% Triton X-100, 0.25% SDS, 0.08% 2-Mercaptoethanol, 1mM EDTA, 10mM KCl) to fully resuspend the precipitate, centrifuge at 4°C, 500g non-fixed angle for 5 minutes in a large refrigerated centrifuge, and carefully discard the supernatant. Repeat this step twice. Pay attention to the state of the supernatant liquid after centrifugation. If there is still a lot of turbid material, add this washing step 1-2 times. Through multiple washing and low-speed centrifugation, remove the complex cellular inclusions in the plant economic organs as much as possible while retaining the chromatin.

[0043] 2.3 Take 15 ml of washing buffer Z3 (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.1% Tween-20, 1% Triton X-100) to fully resuspend the precipitate, centrifuge at 4°C, 500g for 5 minutes at a non-fixed angle, and carefully discard the supernatant to remove alcohol, SDS and other substances in buffer Z2.

[0044] 3. Take 2-4mL of washing buffer Z4 (50mM HEPES, 150mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate, 0.15% SDS, 1x protease inhibitor), fully resuspend the precipitate, and transfer it to a 15mL centrifuge tube. This step is to concentrate the chromatin and increase the chromatin concentration in the solution.

[0045] 4. Use ultrasound to fragment chromatin. The present invention abandons the step of extracting the cell nucleus. The material that has not completely released the cell nucleus can further release chromatin during the ultrasound process, which greatly improves the efficiency of obtaining fragmented chromatin. Therefore, this step has two rounds of ultrasound process to ensure that the chromatin is fully fragmented. After the first round of ultrasound, centrifuge at 10,000g for 10 minutes at 4°C, carefully remove the supernatant and place it in a new centrifuge tube for a second round of ultrasound (each round of ultrasound conditions is Bioruptor: 30s on / 50soff; 32 cycles). The chromatin solution after ultrasonic fragmentation is centrifuged again, centrifuged at 10,000g for 10 minutes at 4°C, and the clarified middle layer liquid is transferred to a new centrifuge tube (and be careful to avoid substances such as oils and fats in the upper layer and precipitates in the lower layer) to concentrate the chromatin.

[0046] 5. Antibody incubation step: Take 80-120uL protein G magnetic beads and wash them twice with 1xPBST (1x PBS with 0.1% Tween 20) with the help of a magnetic stand, and add 120-180uL 1x PBST to fully resuspend the magnetic beads, add 5ug antibody, and incubate at 4°C, 10rpm for 7-9h.

[0047] 6. Immunoprecipitation step: Wash the incubated Protein G-antibody complex twice with 1xPBST, add the fragmented chromatin solution to the magnetic beads, and rotate and incubate for binding at 4°C and 10rpm for 12-18h. This step is for the antibody to capture the specific target binding protein. For different target proteins, their proportions and binding efficiencies in chromatin are different. Therefore, for highly enriched sites such as histone modifications, the amount of antibodies and magnetic beads used can be appropriately reduced, or the time for immunoprecipitation can be reduced. However, for transcription factors or chromatin enzymes, the degree of binding in chromatin is low and there are few binding sites, so the amount of antibodies and magnetic beads should be appropriately increased, and the time for immunoprecipitation should be increased.

[0048] 7. Cleaning of Non-specific Binding Steps

[0049] 7.1 Place the magnetic beads on a magnetic rack and remove the supernatant. Add 1 mL of pre-cooled low-salt washing buffer (50 mM HEPES-KOH, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS) to fully resuspend the magnetic beads and rotate them at 4°C and 10 rpm for 5 minutes. Repeat this step once.

[0050] 7.2 Place the magnetic beads on a magnetic rack and remove the supernatant. Add 1 mL of pre-cooled high-salt washing buffer (50 mM HEPES-KOH, 350 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS) to fully resuspend the magnetic beads and rotate them at 4°C and 10 rpm for 5 minutes. Repeat this step once.

[0051] 7.3 Place the magnetic beads on a magnetic rack and remove the supernatant. Add 1 mL of pre-cooled ChIP wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP-40, 1 mM EDTA, 0.1% sodium deoxycholate) to fully resuspend the magnetic beads and rotate them at 4°C and 10 rpm for 5 minutes. Repeat this step once.

[0052] 7.4 Place the magnetic beads on a magnetic rack, remove the supernatant, add 1 mL of pre-cooled TE buffer to fully resuspend the magnetic beads, and rotate at 4°C, 10 rpm for 5 minutes.

[0053] 8. Place the magnetic beads on a magnetic rack and remove the supernatant. Add 100uL elution buffer (50mM Tris-HCl pH7.5, 10mM EDTA, 1% SDS) and fully resuspend the magnetic beads in a 2mL centrifuge tube. Place the centrifuge tube in a metal bath and elute at 900rpm for 15min at 65°C. Place the magnetic beads on a magnetic rack, carefully remove the supernatant into a new centrifuge tube, and further elute the magnetic beads with 200ul of water and transfer them to a new centrifuge tube.

[0054] 9. DNA-protein cross-linking: Add 6uL of 5M NaCl and 5uL of proteinase K to 300uL of eluted supernatant, and incubate the centrifuge tube at 55°C for 6h or overnight.

[0055] 10. Add 300uL of phenol: chloroform: isoamyl alcohol and mix thoroughly by pipetting. Centrifuge at 12,000rpm for 5min, carefully take the supernatant into a new centrifuge tube, add 340uL of isopropanol, 2uL of Gly Blue, and 30uL of sodium acetate. Freeze the centrifuge tube at -80℃ for 30min, then centrifuge at 4℃ at 12,000rpm for 25min. Carefully discard the supernatant, wash the precipitate twice with pre-cooled 75% ethanol, dissolve and measure the concentration of ChIP-DNA.

[0056] 11.ChIP-seq library construction

[0057] 11.1 Take out 1-50 ng of ChIP-DNA, use ultrasound to fragment the DNA into 200-300 bp, refer to NEB's DNA library preparation kit to perform end filling, sequencing adapter addition, and PCR amplification for 6-11 cycles.

[0058] 11.2 Use VAHTS DNA Clean Beads to sort out the 300-500 bp fragments in the library.

[0059] 12. Perform high-throughput sequencing on the corresponding ChIP-seq library and compare it with the reference genome of the corresponding species to evaluate the data quality of the obtained ChIP-seq library.

[0060] Example 2

[0061] 1. Introduction to the new aChIP experimental method

[0062] like Figure 1 As shown in the figure, the aChIP method abandons the cell nucleus extraction process of conventional ChIP-seq. The material after liquid nitrogen grinding is directly washed with buffer Z1 / 2 / 3 to remove the complex cell inclusions in the plant economic organs as much as possible. Then, buffer Z4 is used to condense the fragmented chromatin, ultrasonically fragmented, and immunoprecipitated, and then eluted and constructed to obtain a high-quality ChIP-seq library.

[0063] 2. The new aChIP assay yielded better data than other methods in 50-day-old Brassica napus seeds

[0064] like Figure 2 As shown in the figure, compared with eChIP (enhanced ChIP) and rChIP (conventional ChIP), the aChIP experimental method has a high efficiency in chromatin acquisition in the immunoprecipitation step and has the highest ChIP-DNA capture efficiency (this is related to the aChIP method's higher chromatin acquisition efficiency, cleaner and higher concentration of fragmented chromatin). At the same time, the results of partial genome position screenshots and FRiP (the fraction of reads in the peak) show that only the aChIP method can produce high-quality ChIP-seq data in 50-day-old Brassica napus seeds.

[0065] 3. Application of aChIP method in 10 different economically important organs of plants

[0066] like Figure 3As shown, the present invention tested the universality of the new aChIP method in 9 other important economic organs of plants. These include: soybean seeds, corn seeds, rice seeds, carnation flowers, potato tubers, sugarcane stalks, tomato fruits, orange peels and pulps. The present invention can produce high-quality ChIP-seq data in these 10 representative important economic organs of plants, showing enrichment of significant peaks and low noise.

[0067] 4. The data quality produced by the aChIP method in important economic organs of plants is significantly higher than that of published data

[0068] like Figure 4 As shown, the present invention compares the quality differences of potato tuber and tomato fruit data produced by aChIP with the published data of the corresponding materials. The results show that the peak shape and noise performance of aChIP data are significantly better than those of the published data.

[0069] 5. aChIP method can produce high-quality data in transcription factors, DNA binding proteins, chromatin enzymes and RNAPII

[0070] like Figure 5 As shown, the present invention tests the superiority of the aChIP method in materials such as transcription factors, DNA binding proteins and chromatin enzymes, which are difficult to obtain high-quality data using conventional methods. The results show that aChIP can produce high-quality data in transcription factor CAMTA3, DNA binding protein MBD7, and DNA demethylase DME, and the experimental results are better than other experimental methods or published data. At the same time, the present invention tests the applicability of the aChIP method to RNAPII in Brassica napus seeds, and also proves that only the aChIP method can produce high-quality data.

[0071] 6. aChIP method can identify more low-enriched peaks in conventional materials

[0072] like Figure 6 As shown, the present invention tested the versatility of the aChIP method in young leaves of Brassica napus and rice seedlings, and found that aChIP can also produce high-quality data (FRiP is higher than 70%). At the same time, the present invention proves that in conventional materials such as young leaves and seedlings, aChIP can identify more low-enriched peaks compared to other experimental methods, and these peaks are significantly higher than the control, and are enriched in the promoter region, similar to the H3K4me3 modification pattern at other sites. This may be due to the higher concentration and higher purity of fragmented chromatin in the aChIP method in the immunoprecipitation step, which will help to improve the enrichment of target proteins at low-enriched sites. The identification of more peaks helps in the detailed analysis of the subject and the accuracy of the conclusions derived, greatly improving the credibility of the experimental results.

[0073] 7. The data produced by the aChIP method in conventional materials is significantly better than the published CUT&Tag data

[0074] like Figure 7 As shown, the aChIP data produced by the present invention in young leaves of Brassica napus and rice seedlings are significantly better than the published CUT&Tag data in terms of indicators such as the number of peaks, peak shape and noise level.

Claims

1. A ChIP-seq method for plant economic organs, characterized in that: The method comprises the following steps: (1) collecting materials and performing formaldehyde cross-linking and fixation; (2) Add washing buffer Z1, rotate at 4°C, 10 rpm for 10 min, then centrifuge at 4°C, 500 g for 5 min, and remove the supernatant; add buffer Z2 to resuspend the precipitate, centrifuge at 4°C, 500 g for 5 min, and remove the supernatant. Repeat this step twice; add buffer Z3 to resuspend the precipitate, and centrifuge at 4°C, 500 g for 5 min. Use low-speed centrifugation instead of nylon membrane filtration to remove the complex cellular contents in important economic organs of plants; (3) Add buffer Z4 to resuspend the pellet; (4) Use ultrasound to fragment chromatin: perform two rounds of ultrasound. After the first round of ultrasound, centrifuge at 10,000 g for 10 min at 4°C, and take the supernatant into a new centrifuge tube for the second round of ultrasound. The ultrasound conditions for each round are 30 s on / 50 s off, and 32 cycles are performed. The chromatin solution after ultrasound is centrifuged again at 10,000 g for 10 min at 4°C, and the clarified intermediate layer liquid is transferred to a new centrifuge tube. (5) The fragmented chromatin is sequentially incubated with antibodies, immunoprecipitated, nonspecifically bound, decrosslinked DNA-protein, purified, and sequenced; Wherein, the buffer Z1 includes: 20-100 mM HEPES, 100-300 mM NaCl, 0.2-2 mM EDTA, 0.5-2% Triton X-100, 0.05-0.5% sodium deoxycholate, 0.1-0.5% SDS, 1x protease inhibitor; The buffer Z2 includes: 1xPBS, 50-200 mM NaCl, 8-18.8% isohexanediol, 0.1-1% TritonX-100, 0.1-2% SDS, 0.01-0.2% β-mercaptoethanol, 0.1-2 mM EDTA, 1-20 mM KCl; The buffer Z3 includes: 20-100 mM HEPES, 100-300 mM NaCl, 0.1-2 mM EDTA, 0.5-2% Tween-20, 0.5-2% Triton X-100; The buffer Z4 includes: 20-100 mM HEPES, 100-300 mM NaCl, 0.1-2 mM EDTA, 0.5-2% Triton X-100, 0.01-0.2% sodium deoxycholate, 0.01-0.3% SDS, 1x protease inhibitor.

2. The method according to claim 1, characterized in that: The step (1) is to chop the collected material into pieces, place it in a 1xPBS solution, and perform cross-linking and fixation with formaldehyde in a vacuum environment. The gas is evacuated 2-3 times and stirred to mix well. After ensuring that the material is fully cross-linked and fixed, the material is placed in a glycine solution to terminate the cross-linking reaction.

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