Efficient plant ChIPmentation method based on chromatin preenrichment

Abstract

The invention belongs to the technical field of high-throughput experiments of molecular biology, and relates to an efficient plant ChIPmentation method based on chromatin preenrichment. Comprising the following steps: carrying out formaldehyde crosslinking on a plant tissue sample, extracting a cell nucleus, carrying out ultrasonic fragmentation on DNA, precipitating a DNA-target protein compound, dissolving and precipitating by using a low-salt solution, fixing the DNA-target protein compound on magnetic beads by using a specific target protein antibody, fragmenting the DNA by using Tn5 transposase, adding sequencing joints at two ends of the fragmented DNA, and carrying out sequencing on the DNA-target protein compound. DNA is directly released from the magnetic beads through decrosslinking, DNA purification is carried out, a sequencing library is constructed, and sequencing is completed. The core innovation of the invention lies in that the combination efficiency of the antibody magnetic beads and the target protein is significantly improved by precipitating a DNA-target protein compound and dissolving the DNA-target protein compound in a low-salt solution, so that the whole genome accurate positioning research of plant histone modification or a transcription factor and DNA combination mode is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology high-throughput experiments, and specifically relates to an efficient method for separating plant histones or transcription factors and their bound DNA, which is suitable for ChIP-seq sample preparation. Background Art

[0002] High-throughput sequencing technologies such as ChIP-seq (Chromatin Immunoprecipitation Sequencing) have played a key role in revealing genomic regulatory mechanisms. However, ChIP-seq analysis of plant tissue samples, such as the study of histone modification H3K4me3 or transcription factors, faces technical challenges and limitations. Traditional methods have limited enrichment of target protein and DNA complexes, and are often accompanied by more interference from other proteins, resulting in a low signal-to-noise ratio.

[0003] For example, the invention patent application with application publication number CN 118109571 A discloses a transcription factor ChIP-seq method suitable for plant application, including the following steps: (1) fixation of leaf material; (2) extraction of cell nuclei and DNA shearing (using nuclear lysis buffer for DNA shearing); (3) DNA co-precipitation (using label antibodies and sheared DNA fragments for immunoprecipitation reaction); (4) Tn5 enzyme reaction; (5) cross-linking removal reaction. Although this method realizes the application of ChIP-seq technology in plant leaves, the sequencing results of this method have a high signal-to-noise ratio and the sequencing data quality is limited.

[0004] Therefore, it is crucial to develop a ChIP-seq method suitable for enriching DNA and target protein complexes in plant tissues, so as to detect and quantify the distribution of target proteins such as histone modifications or transcription factor binding sites in samples with high precision. Summary of the invention

[0005] The present invention aims at the problem that the ChIP-seq technology in the prior art is easy to enrich foreign proteins and has a high signal-to-noise ratio, and provides a high-efficiency plant ChIPmentation based on chromatin pre-enrichment, namely ChIP with sequencing library preparation by Tn5 transposase (tagmentation). The method has high sensitivity and high efficiency, can effectively reduce the interference of foreign proteins, and can significantly improve the signal-to-noise ratio by enriching DNA and target protein complexes, and accurately detect and quantify the distribution of target proteins such as histone modifications or transcription factor binding sites. Through the application of the present invention, it is possible to deeply explore the change pattern of histone modification or transcription factor binding regulation in plants under different tissues and growth conditions, such as the relationship between plant tissue histone modification H3K4me3 and gene expression regulation, thereby revealing its fine mechanism in gene expression regulation.

[0006] The present invention can be achieved through the following technical solutions: An efficient plant ChIPmentation method based on chromatin pre-enrichment, comprising: First, the plant tissue samples are cross-linked with formaldehyde, and then the cell nuclei of the plant tissue are extracted. The DNA is then ultrasonically fragmented and the DNA-target protein complex is precipitated. The precipitate is dissolved with a small volume of low-salt solution, and the DNA-target protein complex is fixed on magnetic beads using specific target protein antibodies. The DNA is fragmented using Tn5 transposase and sequencing adapters are added to both ends of the fragmented DNA. The DNA is then released from the magnetic beads by resolving the cross-links. After DNA purification, a sequencing library is constructed and sequencing is completed.

[0007] The specific steps are as follows: (1) Formaldehyde cross-linking of plant tissue samples: The purpose of this step is to form a reversible covalent bond between proteins (such as histones or transcription factors) and DNA in plant tissues. The specific operations are: Take the plant tissue and immerse it in a cross-linking buffer containing 1% w / v formaldehyde, and evacuate it. Add the cross-linking termination buffer and evacuate it. In this step, the cross-linking buffer and the termination buffer can be used to immerse the plant tissue.

[0008] The solute composition of the cross-linking buffer is: 1×PBS, 1% w / v paraformaldehyde, 1 micromole per milliliter PMSF, which are dissolved to a fixed volume with sterile deionized water; wherein PMSF is pre-dissolved with dimethyl sulfoxide (DMSO).

[0009] The solute composition of the cross-link termination buffer is: 500 micromol / ml glycine and 500 micromol / ml Tris-HCl at pH 7.5, which are dissolved to a fixed volume with sterile deionized water.

[0010] (2) Extraction of cell nuclei from plant tissues: The plant tissue samples after formaldehyde cross-linking were ground, and then nuclear extraction solution was added to extract the nuclei at 0°C; then the samples were passed through a 100-micron cell sieve and a 40-micron cell sieve to remove cell debris other than the nuclei, and then washed with nuclear extraction solution until the nuclear precipitate was grayish white. 10 ml of nuclear extraction solution was added for every 0.2 g of plant tissue.

[0011] The solute composition of the cell nuclear extract is: 0.6 mol / L sorbitol, 10 micromol / mL Tris-HCl with pH 8.0, 1 micromol / mL EDTA, 0.5% v / v Triton X-100 and 1 micromol / mL PMSF, which are dissolved to a fixed volume with sterile deionized water; wherein PMSF is pre-dissolved with dimethyl sulfoxide.

[0012] (3) Ultrasonic fragmentation of DNA: After resuspending the cell nucleus precipitate obtained in step (2) in an ultrasonic buffer, ultrasonication is performed to fragment the DNA. A medium salt buffer is added to the ultrasonicated solution to dissolve the DNA to obtain a DNA solution, and the DNA solution is pre-cleaned with magnetic beads.

[0013] The solute composition of the ultrasonic buffer is: 10 micromoles per milliliter Tris-HCl at pH 8.0, 1 micromoles per milliliter EDTA, 0.15% w / v sodium dodecyl sulfate (SDS), 1× protease inhibitor and 1 micromoles per milliliter PMSF, which are dissolved to a fixed volume with sterile deionized water; wherein PMSF is pre-dissolved with dimethyl sulfoxide. The protease inhibitor used is a broad-spectrum protease inhibitor, which is a commercially available product; the PMSF used is an inhibitor for serine proteases; the use of two protease inhibitors together can enhance the protease inhibition effect.

[0014] The solute composition of the medium salt buffer is: 300 micromol / ml NaCl, 2% v / v Triton X-100, 1× protease inhibitor, 10 micromol / ml Tris-HCl with pH 8.0 and 1 micromol / ml EDTA, which are dissolved to a fixed volume with sterile deionized water.

[0015] (4) Precipitation of DNA-target protein complex: Add glycogen, sodium acetate and pre-cooled ethanol to the DNA solution obtained in step (3), let it stand at -20°C to precipitate the DNA-target protein complex, dissolve the precipitate with a low salt solution after centrifugation, and remove impurities to obtain a DNA-target protein complex solution. The pre-cooled ethanol is anhydrous ethanol placed at -20°C for 20 minutes. As an embodiment, the concentration of glycogen is 20 mg / ml, the concentration of sodium acetate is 0.3 μmol / ml, and the volume ratio of glycogen, sodium acetate and pre-cooled ethanol is 1:50:1500.

[0016] The DNA-target protein complex precipitate obtained from each gram of plant tissue was dissolved in 125 microliters of low salt solution.

[0017] The solute composition of the low-salt solution is: 150 micromol / ml NaCl, 0.1% v / v Triton X-100, 1× protease inhibitor, 10 micromol / ml Tris-HCl with pH 8.0 and 1 micromol / ml EDTA, which are dissolved and fixed to volume with sterile deionized water. The protease inhibitor used is a broad-spectrum protease inhibitor, which is a commercially available product.

[0018] (5) Magnetic beads bind to specific target protein antibodies and target proteins: After washing and resuspending the magnetic beads with a low salt solution, add the specific target protein antibody and incubate. Then mix with the DNA-target protein complex solution obtained in step (4) and incubate. Then wash the incubated magnetic beads with a low salt solution, a high salt solution, and a Tween solution in sequence.

[0019] The composition of the low-salt solution is the same as that of step (4); The solute composition of the high salt solution is: 0.5 mol / L NaCl, 10 micromol / mL Tris-HCl with pH 8.0, 1 micromol / mL EDTA, 0.5% v / v Triton X-100, 0.5% DOC, and 1 micromol / mL PMSF, which are dissolved to a fixed volume with sterile deionized water; wherein PMSF is pre-dissolved with dimethyl sulfoxide.

[0020] The solute composition of the Tween solution is: 10 micromol / ml Tris-HCl with a pH of 8.0, 1 micromol / ml EDTA and 0.1% v / v Tween 20.

[0021] (6) Tn5 transposase-labeled magnetic beads-bound DNA-target protein complex (fragment the DNA with Tn5 transposase and add sequencing adapters to both ends of the fragmented DNA): Wash and resuspend the magnetic beads with Tn5 buffer, add Tn5 transposase and sequencing adapter, mix well, and incubate. Wash the magnetic beads with high salt solution without phenylmethylsulfonyl fluoride (PMSF) and Tween buffer.

[0022] The solute composition of the Tn5 buffer is: 20 micromoles per milliliter of Tris-HCl at pH 8.0 and 10 micromoles per milliliter of MgCl2, which are dissolved to a fixed volume with sterile deionized water.

[0023] Before use, the sequencing adapters were pre-incubated with Tn5 transposase at 25°C for 1 hour.

[0024] (7) De-crosslinking: Add cross-linking decoupling buffer to decoupling, and the decoupling conditions are: incubation at 55°C for 30 minutes and incubation at 63°C for 4 hours.

[0025] The solute composition of the cross-linking removal buffer is: 10 micromol / ml Tris-HCl with a pH of 8.0, 5 micromol / ml EDTA, 300 micromol / ml NaCl, 0.2% w / v SDS, 1 micromol / ml oligonucleotide, and 2% v / v proteinase K, which is dissolved to a fixed volume with sterile deionized water.

[0026] (8) DNA purification and library construction: Purify the DNA after cross-linking in (7), add downstream primers and PCR mixture to amplify and construct a library, remove primer dimers with magnetic beads, and use Illumina or other platforms for high-throughput sequencing to obtain sequencing data. Use bioinformatics tools to analyze the sequencing data and identify the genomic location and pattern of histone modifications.

[0027] The beneficial effects of the present invention are: The method of the present invention is an improvement on traditional ChIP-seq. This method removes the sequencing background that may interfere with the sequencing results by first precipitating the sheared DNA and then dissolving it with a small volume of low-salt solution, thereby increasing the magnetic bead binding efficiency and enhancing the target fragment signal.

[0028] The present invention can effectively reduce the background interference of foreign proteins by enriching DNA and target protein complexes, detect and quantify the distribution of target proteins such as histone modifications or transcription factor binding sites with high sensitivity and efficiency, and observe the whole genome modification sites of histones such as H3K4me3 in plant tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1:Comparison of data between the method of the present invention and the traditional ChIP-seq technology on histone H3K4me3 experiments. (A) The enrichment of the method of the present invention and the traditional ChIP-seq technology in specific genomic regions; (B) Demonstration that the signal-to-noise ratio of the data produced by the method of the present invention in histone H3K4me3 is better than that of the traditional ChIP-seq method.

[0030] Figure 2 :(A) Enrichment heat map of transcription start sites where the method of the present invention identified more low-enrichment peaks in the histone H3K4me3 assay;(B) Screenshot of partial genomic positions where the method of the present invention identified more low-enrichment peaks in histone H3K4me3;(C) Comparison of partial genomic positions where the method of the present invention had better resolution in histone H3K4me3 than traditional ChIP-seq technology.

[0031] In the figure, Our method represents the method of the present invention (Example 1), and SRR5748812 represents the traditional ChIP-seq technology (Example 2). DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention will be clearly described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The histone H3K4me3 modification involved in the examples is widely involved in the regulation of gene transcription in plants, and its dynamic changes in specific tissues and growth stages have an important impact on the growth and development of plants.

[0034] In this embodiment, the plant tissue sample is first cross-linked with formaldehyde, and then the cell nucleus of the plant tissue is extracted, followed by DNA fragmentation, precipitation of DNA-target protein complex, and then the precipitation is dissolved with a small volume of low-salt solution, and the DNA-target protein complex is fixed on magnetic beads by specific target protein antibodies, and the DNA is fragmented with efficient and stable TS-Tn5 and sequencing adapters are added to both ends of the fragmented DNA, and then the DNA is released from the magnetic beads by de-crosslinking, and the library is constructed after DNA purification, and high-throughput sequencing is performed using Illumina or other platforms to obtain sequencing data. Then, the sequencing data is analyzed using bioinformatics tools to identify the genomic location and pattern of histone modifications.

[0035] The technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0036] Example 1 An efficient plant ChIPmentation method based on chromatin pre-enrichment comprises the following steps: (1) Formaldehyde cross-linking of corn leaf samples: Take 2g of corn leaves and immerse them in cross-linking buffer (1×PBS, 1% w / v formaldehyde, 1 micromol / ml PMSF), evacuate to the maximum, let it stand for 5 minutes, let it stand for 5 minutes after venting, repeat three times. Pour out the cross-linking buffer and replace it with termination cross-linking buffer (500 micromol / ml glycine and 500 micromol / ml Tris-HCl pH 7.5), evacuate to the maximum, let it stand for 5 minutes, let it stand for 5 minutes after venting.

[0037] (2) Extraction of corn leaf nuclei: Wipe dry the liquid on the leaf surface in step (1), quickly plunge into liquid nitrogen, and grind thoroughly with a mortar. Dissolve the ground sample in a 50 ml centrifuge tube with a nuclear extraction solution (0.6 mol / L sorbitol, 10 μmol / mL Tris-HCl pH 8.0, 1 μmol / mL EDTA, 0.5% v / v Triton X-100, and 1 μmol / mL PMSF) (add 10 ml of nuclear extraction solution for every 0.2 g corn leaf powder), then place in an ice box and shake on a shaker for 10 minutes. Pass all the liquid through a 100 μm cell sieve, collect the liquid in a new 50 ml centrifuge tube, and centrifuge at 3000 g at 4°C for 10 minutes. Pour off the upper liquid, resuspend with 2 ml of nuclear extraction solution, pass the resuspended liquid through a 40 μm cell sieve, collect the liquid in a 2 ml centrifuge tube, and centrifuge at 3000 g at 4°C for 5 minutes. Observe the color of the nuclear precipitate until it is washed with nuclear extraction solution to grayish white.

[0038] (3) Ultrasonic fragmentation of DNA: Resuspend the cell nuclei obtained in step (2) in 250 μl of sonication buffer (10 μmol / ml Tris-HCl pH 8.0, 1 μmol / ml EDTA, 0.15% w / v sodium dodecyl sulfate (SDS), 1× protease inhibitor cocktail, and 1 μmol / ml PMSF), and sonicate using a sonicator (30 seconds on / 30 seconds off) for 5 cycles, for a total of three times. Add 250 μl of medium salt buffer (300 μmol / ml NaCl, 2% v / v Triton X-100, 1× protease inhibitor cocktail, 10 μmol / ml Tris-HCl pH 8.0, and 1 μmol / ml EDTA) to the sonicated solution, rotate and mix at 4°C for 10 minutes, centrifuge at 20,000 g at 4°C for 5 minutes, aspirate the supernatant, add pre-cleaned magnetic beads, and rotate and mix at 4°C for 2 hours. Use a magnet to remove the magnetic beads, centrifuge at 20,000 g at 4°C for 5 minutes, and carefully aspirate the supernatant into a new 2 ml centrifuge tube. The 1× protease inhibitor cocktail used refers to: EDTA-free cOmplete™ protease inhibitor cocktail (cOmplete ULTRATablets, EDTA-free, Roche, Catalog Number: 06538304001) and EDTA-free Pierce™ protease inhibitor cocktail (Protease Inhibitor Mini Tablets, Thermo, Catalog Number: A32953) are prepared into 10× stock solution and 100× stock solution respectively according to the product instructions; when preparing the sonication buffer (or medium salt buffer), 100 μl of 10× EDTA-free cOmplete™ protease inhibitor cocktail and 10 μl of 100× EDTA-free Pierce™ protease inhibitor cocktail are required for each milliliter of sonication buffer (or medium salt buffer).

[0039] (4) Precipitation of DNA-target protein complex: Add 1 μl of 20 mg / ml glycogen, 50 μl of 0.3 mol / l sodium acetate and 1500 μl of pre-cooled ethanol to the sample in step (3), mix thoroughly and let stand at -20°C for half an hour. Centrifuge at maximum speed (20 kg) for 20 minutes at 4°C, discard the supernatant, and dissolve the precipitate with 250 μl of low salt solution (0.15 mol / l NaCl, 10 μl / ml Tris-HCl pH 8.0, 1 μl / ml EDTA, 0.1% v / v Triton X-100, 1× protease inhibitor cocktail and 1 μl / ml PMSF) to obtain a DNA-target protein complex solution. The 1× protease inhibitor cocktail used was prepared by preparing 10× and 100× stock solutions of EDTA-free cOmplete™ protease inhibitor cocktail (cOmplete ULTRA Tablets, EDTA-free, Roche, Catalog Number: 06538304001) and EDTA-free Pierce™ protease inhibitor cocktail (Protease Inhibitor MiniTablets, Thermo, Catalog Number: A32953), respectively, according to the product instructions; when preparing the low salt solution, 100 μl of 10× EDTA-free cOmplete™ protease inhibitor cocktail and 10 μl of 100× EDTA-free Pierce™ protease inhibitor cocktail were required per milliliter of low salt solution.

[0040] (5) Magnetic beads bind to specific target protein antibodies: The magnetic beads were washed three times with low salt solution, resuspended with 100 μl of low salt solution, and 1 μl of H3K4Me3 antibody (Upstate®, Catalog Number: 04-745) was added, and incubated with rotation at 4°C for 6 hours to obtain a magnetic bead suspension.

[0041] (6) Antibody-carrying magnetic beads bind to target proteins: Add the magnetic bead suspension to the DNA-target protein complex solution in step (4) and incubate with rotation at 4°C for 12 hours.

[0042] (7) Magnetic bead cleaning: Recover the beads on a pre-cooled (pre-cooled on ice) magnetic stand, resuspend in 500 μL of ice-cold low salt solution, and wash twice for 5 minutes at 4°C. Recover the beads on a pre-cooled magnetic stand, wash twice with 500 μL of ice-cold high salt solution (0.5 M NaCl, 10 μL Tris-HCl pH 8.0, 1 μL EDTA, 0.5% v / v Triton X-100, 0.5% w / v sodium deoxycholate (DOC), 1 μL PMSF), rotate for 5 minutes at 4°C each time. Wash with 150 μL of ice-cold Tween solution (10 μL Tris-HCl pH 8.0, 1 μL EDTA, and 0.1% v / v Tween 20), and let stand on ice for 2 minutes.

[0043] (8) TS-Tn5 transposase fragments the DNA and adds sequencing adapters to both ends of the fragmented DNA: TS-Tn5 transposase is a modified Tn5 transposase, which is a reference (Tu, X., Mejía-Guerra, MK, Valdes Franco, JA et al. Reconstructing the maize leaf regulatory network using ChIP-seq data of 104 transcription factors. Nat Commun 11, 5089 (2020). https: / / doi.org / 10.1038 / s41467-020-18832-8) was transformed.

[0044] Wash the beads once with 150 μl Tn5 buffer (20 μmol / ml Tris-HCl pH 8.0 and 10 μmol / ml MgCl2) without N,N-dimethylformamide (DMF), then transfer to a new PCR tube, resuspend the beads with 50 μl Tn5 buffer, add TS-Tn5 transposase (1OD) and sequencing adapters, and mix thoroughly (before use, the sequencing adapters and TS-Tn5 transposase were pre-incubated at 25°C for 1 hour). Incubate at 37°C for 45 minutes. Wash the beads twice with 150 μl high salt solution, and once with high salt solution without phenylmethylsulfonyl fluoride (PMSF) and Tween buffer.

[0045] The sequencing adapter used adopts the standardized P7 / P5 adapter sequence of the Illumina sequencing platform, and the sequence is as follows: Illumina P5 Sequence: 5'-AATGATACGGCGACCACCGAGATCTACAC-3'; Illumina P7 Sequence: 5'-ATCTCGTATGCCGTCTTCTGCTTG-3'.

[0046] (9) De-crosslinking: Add 50 μL of cross-linking reversal buffer (10 μM Tris-HCl pH 8.0, 5 μM EDTA, 300 μM NaCl, 0.2% w / v SDS, 1 μM oligonucleotide (Shanghai Sangon Biotechnology Co., Ltd., Catalog No.: 500056), 2% v / v proteinase K (Shanghai Sangon Biotechnology Co., Ltd., Catalog No.: B600169)), and use a PCR instrument to reverse the cross-linking, setting the program: 55°C for 30 minutes, 63°C for 4 hours.

[0047] (10) DNA purification and library construction: Use the zymoDNA purification kit (Zymoresearch, DNA Clean-up and Concentration kit, catalog number: D4033) to purify the DNA in (9), add downstream primers (i5 index: GCGATCTA, i7 index: TAAGGCGA) and PCR mixture to amplify and construct a library, then use AMPure magnetic beads to remove primer dimers, use Illumina or other platforms for high-throughput sequencing, and obtain sequencing data. Use bioinformatics tools to analyze the sequencing data and identify the genomic location and pattern of histone modifications.

[0048] The PCR amplification system used (60 μl) was: 0.5 μl Q5 DNA polymerase, 12 μl 1× Q5 buffer (NEBQ5 ® High-Fidelity DNA Polymerase, Catalog No.: M0491V), 1.2 μl 10 μmol / ml oligonucleotide (Shanghai Sangon Biotechnology Co., Ltd., Catalog No.: 500056), 40.3 μl purified DNA solution, 3 μl 0.5 μmol / ml i5 index, 3 μl 0.5 μmol / ml i7 index.

[0049] Example 2 Comparative Example Using the traditional ChIP-seq method (see Tu, X., Mejía-Guerra, MK, Valdes Franco, JA et al.Reconstructing the maize leaf regulatory network using ChIP-seqdata of 104 transcription factors. Nat Commun 11, 5089 (2020). https: / / doi.org / 10.1038 / s41467-020-18832-8) for chromatin enrichment. The plant tissue sample used in this example was also 2 g of corn leaves.

[0050] The enrichment of the method of the present invention (Example 1) and the traditional ChIP-seq method (Example 2) in a specific genomic region and the signal-to-noise ratio of the data were compared. The results are as follows: Figure 1 As shown, when using the same amount of sequencing data, the enrichment signal at the transcription start site of the method of the present invention is significantly stronger than the background noise, which will effectively improve data utilization and downstream analysis accuracy.

[0051] according to Figure 2 It can be concluded that the method of the present invention identified more transcription start sites with low enrichment peaks in the histone H3K4me3 assay ( Figure 2 A), the part of the genome where the method of the present invention identified more low-enrichment peaks in histone H3K4me3 ( Figure 2 B) The proposed method has better resolution than traditional ChIP-seq technology in histone H3K4me3 ( Figure 2 C).

[0052] In general, the present invention can effectively reduce the background interference of foreign proteins, and detect and quantify the distribution of target proteins such as histone modifications or transcription factor binding sites with high sensitivity and high resolution.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An efficient plant ChIPmentation method based on chromatin pre-enrichment, characterized in that: include: First, the plant tissue samples are cross-linked with formaldehyde, and then the cell nuclei of the plant tissues are extracted. Then, the DNA is fragmented by ultrasound, the DNA-target protein complex is precipitated, and the precipitate is dissolved with a low-salt solution. The DNA-target protein complex is fixed on magnetic beads using a specific target protein antibody, and the DNA is fragmented using Tn5 transposase and sequencing adapters are added to both ends of the fragmented DNA. The DNA is then released from the magnetic beads by de-crosslinking, and the DNA is purified to construct a sequencing library and complete sequencing; The solute composition of the low salt solution is: 150 micromol / ml NaCl, 0.1% v / v Triton X-100, 1× protease inhibitor, 10 micromol / ml Tris-HCl with pH 8.0, and 1 micromol / ml EDTA.

2. The efficient plant ChIPmentation method based on chromatin pre-enrichment according to claim 1, characterized in that: The method of performing formaldehyde cross-linking on the plant tissue sample comprises: immersing the plant tissue in a cross-linking buffer solution containing formaldehyde and evacuating the solution; and adding a cross-linking termination buffer solution and evacuating the solution.

3. The efficient plant ChIPmentation method based on chromatin pre-enrichment according to claim 1, characterized in that: The method for extracting cell nuclei from plant tissues comprises: grinding the plant tissue sample after formaldehyde cross-linking, then adding a cell nucleus extracting solution, and extracting the cell nuclei under 0° C.; removing cell debris, and washing the sample with a cell nucleus extracting solution until the cell nuclei are precipitated to be grayish white.

4. The efficient plant ChIPmentation method based on chromatin pre-enrichment according to claim 1, characterized in that: When ultrasonically fragmenting DNA, ultrasonic buffer is added to the nuclear pellet before ultrasonication; The solute composition of the sonication buffer was: 10 μM Tris-HCl, pH 8.0, 1 μM EDTA, 0.15% w / v sodium dodecyl sulfate, 1× protease inhibitor, and 1 μM PMSF.

5. The efficient plant ChIPmentation method based on chromatin pre-enrichment according to claim 1, characterized in that: After the ultrasonic fragmentation of the DNA, the method further includes dissolving the DNA with a medium salt buffer and pre-cleaning the obtained DNA solution with magnetic beads; The solute composition of the medium salt buffer is: 300 micromol / ml NaCl, 2% v / v Triton X-100, 1× protease inhibitor, 10 micromol / ml Tris-HCl at pH 8.0, and 1 micromol / ml EDTA.

6. The efficient plant ChIPmentation method based on chromatin pre-enrichment according to claim 1, characterized in that: The precipitation of the DNA-target protein complex comprises: adding glycogen, sodium acetate and pre-cooled ethanol to the fragmented DNA solution, and centrifuging; The pre-cooled ethanol refers to ethanol that has been placed at -20°C for 20 minutes.

7. The efficient plant ChIPmentation method based on chromatin pre-enrichment according to claim 1, characterized in that: The method uses specific target protein antibodies to fix the DNA-target protein complex on magnetic beads, including: washing and resuspending the magnetic beads with a low salt solution, adding the specific target protein antibodies, and incubating; then mixing with the DNA-target protein complex solution and incubating; and washing the incubated magnetic beads.

8. The efficient plant ChIPmentation method based on chromatin pre-enrichment according to claim 7, characterized in that: The washing of the incubated magnetic beads comprises: washing with a low salt solution, a high salt solution, and a Tween solution in sequence; The solute composition of the high salt solution is: 0.5 mol / L NaCl, 10 μmol / mL Tris-HCl at pH 8.0, 1 μmol / mL EDTA, 0.5% v / v Triton X-100, 0.5% w / v sodium deoxycholate, and 1 μmol / mL PMSF; The solute composition of the Tween solution is: 10 micromol / ml Tris-HCl with a pH of 8.0, 1 micromol / ml EDTA and 0.1% v / v Tween 20.

9. The efficient plant ChIPmentation method based on chromatin pre-enrichment according to claim 1, characterized in that: The method of fragmenting DNA using Tn5 transposase and adding sequencing adapters to both ends of the fragmented DNA includes: washing and resuspending magnetic beads with Tn5 buffer, adding Tn5 transposase and sequencing adapters, mixing, incubating, and washing magnetic beads; The solute composition of the Tn5 buffer is: 20 micromoles per milliliter of Tris-HCl at pH 8.0, 10 micromoles per milliliter of MgCl2, and 15% v / v DMF; The Tn5 transposase and sequencing adapters were pre-incubated at 25°C for 1 hour.

10. The efficient plant ChIPmentation method based on chromatin pre-enrichment according to claim 1, characterized in that: The de-crosslinking comprises: adding a de-crosslinking buffer to the magnetic beads to de-crosslink, and the de-crosslinking conditions are: incubating at 55° C. for 30 minutes and incubating at 63° C. for 4 hours; The solute composition of the cross-linking removal buffer is: 10 micromol / ml Tris-HCl at pH 8.0, 5 micromol / ml EDTA, 300 micromol / ml NaCl, 0.2% w / v SDS, 1 micromol / ml oligonucleotide, and 2% v / v proteinase K.

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