High-resolution promoter interaction fragment targeted capture method suitable for plants

Abstract

Provided is a high-resolution promoter interaction fragment targeted capture method suitable for plants, which comprises: constructing a chromatin conformation capture pre-library; designing an RNA probe reversely complementary to a core promoter sequence; using the RNA probe to capture a promoter interaction fragment in the chromatin conformation capture pre-library; amplifying the library; and performing sequencing to obtain information of the promoter interaction fragment. By means of complementarily pairing hybridization between an RNA and a DNA, the capture method can specifically capture a promoter interaction fragment from a high-resolution chromatin conformation capture library, so as to obtain information of the promoter interaction fragment. Compared with existing capture technologies, the method exhibits greatly improved capture efficiency and has an obvious advantage in searching for distal regulatory elements.

Description

Method for high-resolution targeted capture of promoter interacting fragments suitable for plants TECHNICAL FIELD

[0001] The present application relates to the technical field of chromosome conformation capture, in particular to a method for high-resolution targeted capture of promoter interacting fragments suitable for plants. BACKGROUND

[0002] Chromosome conformation capture technology is a technology for studying the higher-order structure of chromatin, which can analyze the correlation between gene sites that are very far apart in linear distance. In recent years, with the rapid development of the second-generation sequencing technology, high-resolution chromosome conformation capture technology (Hi-C) derived from chromosome conformation capture technology has taken the whole nucleus as the research object to study the correlation between gene sites in the whole genome.

[0003] Promoters play an important regulatory role in gene transcription. High-resolution chromatin conformation capture technology can theoretically capture all interactions within the genome at the same time. However, the information of high-resolution chromatin conformation capture library is very complex. Therefore, it is difficult to achieve the required sequencing depth to obtain promoter-targeted fragments. In mammals, the long-range interaction of promoters and enhancers forms a special chromatin loop structure, which regulates the transcription of the corresponding genes. However, in plants, it is still not possible to identify DNA elements that interact with promoters and affect transcription in the whole genome.

[0004] Some targeted capture technologies have also been reported, mostly based on ChIP-seq technology. However, since no specific protein or histone modification has been found in plants that interacts with promoters, it is not possible to use the above ChIP-seq technology for whole-genome capture. In addition, some other technologies for identifying DNA regulatory elements have also been reported. The study of enhancers in plants mainly relies on the openness of chromatin, and a large number of studies have used a series of such technologies to find many "enhancer candidates" in plants. However, the number of functionally verified ones is very limited, so this type of technology is not the best choice for efficiently finding promoter interacting elements.

[0005] Therefore, it is necessary to develop a method for high-resolution targeted capture of promoter interacting fragments suitable for plants.

[0006] SUMMARY

[0007] The present application aims to provide a method for high-resolution targeted capture of promoter interacting fragments suitable for plants. The promoter interacting fragments are specifically captured from the high-resolution chromatin conformation capture library through complementary pairing and hybridization of RNA and DNA, and then the information of the promoter interacting fragments is obtained. The specific technical solutions are as follows:

[0008] The first aspect of the present application provides a method for high-resolution targeted capture of promoter interaction fragments suitable for plants, comprising: constructing a chromatin conformation capture pre-library; designing RNA probes complementary to the core promoter sequence; capturing promoter interaction fragments in the chromatin conformation capture pre-library using the RNA probes; library amplification; sequencing to obtain promoter interaction fragment information; wherein the step of capturing promoter interaction fragments in the chromatin conformation capture pre-library using the RNA probes comprises: adding RNase inhibitor and the RNA probes to the chromatin conformation capture pre-library, hybridizing for 16-20 hours to obtain a hybridization mixture; adding streptavidin magnetic beads to the hybridization mixture, mixing, incubating with shaking, centrifuging, resuspending the magnetic beads after discarding the supernatant, incubating for 10-20 minutes, vortexing every 5-10 minutes, centrifuging, resuspending the magnetic beads after discarding the supernatant, incubating at 60-70°C for 8-12 minutes; washing 2-4 times, resuspending the magnetic beads after discarding the supernatant, and adding sterile water.

[0009] In an embodiment of the present application, the volume ratio of the chromatin conformation capture pre-library, the RNase inhibitor, and the RNA probes is 3-8:0.3-0.8:1-3; and the volume ratio of the streptavidin magnetic beads to the hybridization mixture is 15:2-4.

[0010] In an embodiment of the present application, the step of library amplification comprises: performing PCR reaction on the promoter interaction fragments captured by the RNA probes; after the PCR reaction is completed, adding DNA purification magnetic beads and incubating for 3-7 minutes; discarding the supernatant after standing for 3-5 minutes, adding 70-90 vol% ethanol and incubating for 20-40 seconds, adding 70-90 vol% ethanol again after discarding the supernatant, incubating for 20-40 seconds, discarding the supernatant, standing for 1-3 minutes; adding ultrapure water for elution and standing for 1-3 minutes; taking the supernatant and sequencing to obtain promoter interaction fragment information.

[0011] In an embodiment of the present application, the step of constructing a plant chromatin conformation capture pre-library comprises: cross-linking the sample to obtain cross-linked material; performing nuclear extraction on the cross-linked material to obtain nuclear extraction material; performing restriction enzyme digestion on the nuclear extraction material to obtain enzyme-digested material; performing sticky end smoothing on the enzyme-digested material using biotin-labeled bases to obtain end-smoothed material; performing intranuclear ligation on the end-smoothed material to obtain intranuclear-ligated material; performing DNA de-cross-linking, extracting DNA, and using ultrasonic waves to break the DNA fragments, sorting, and performing biotin enrichment using magnetic beads to construct a chromatin conformation capture pre-library.

[0012] In an embodiment of the present application, the step of nuclear extraction comprises: resuspending the cross-linked material with NIB buffer, filtering, collecting the filtrate, centrifuging at 3000-5000 rpm for 8-12 minutes at 3-5°C, and discarding the supernatant; resuspending again with NIB buffer, centrifuging at 3000-5000 rpm for 8-12 minutes at 3-5°C, discarding the supernatant, and repeating 1-2 times; resuspending again with NIB buffer, centrifuging at 2000-4000 rcf for 3-7 minutes at 3-5°C, and discarding the supernatant; the NIB buffer comprises: 10-30 mM (mmol / L) 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES), pH 7.5-8.5; 200-300 mM sucrose; 0.5-1.5 mM magnesium chloride; 3-8 mM potassium chloride; 30-50 vol% glycerol; 0.2-0.3 vol% polyethylene glycol octylphenyl ether (Triton X-100); 0.05-0.15 mM benzylsulfonyl fluoride; 0.05-0.15 vol% protease inhibitor; 0.05-0.15 vol% β-mercaptoethanol.

[0013] In an embodiment of the present application, the step of restriction enzyme digestion comprises: resuspending the nuclear extraction material with 1× RE buffer, centrifuging at 2000-4000 rcf for 3-7 minutes at 3-5°C, and discarding the supernatant; resuspending the precipitate with 0.3-0.7 vol% sodium dodecyl sulfate (SDS) solution, incubating at 60-65°C for 3-7 minutes; adding 8-12 vol% Triton X-100, incubating at 35-40°C for 10-20 minutes; adding 10× RE buffer and restriction enzyme, incubating at 35-40°C for 12-16 hours; the 1× RE buffer comprises: 0.005-0.015 M (mol / L) sodium chloride; 4-6 mM Tris-HCl, pH 7.5-8.5; 0.8-1.2 mM magnesium chloride; 0.05-0.15 mM dithiothreitol (DTT); the 10× RE buffer comprises: 0.05-0.15 M sodium chloride; 40-60 mM Tris-HCl, pH 7.5-8.5; 8-12 mM magnesium chloride; 0.5-1.5 mM dithiothreitol.

[0014] In an embodiment of the present application, the step of sticky end filling comprises: incubating the digested material at 60-65°C for 15-25 minutes, and then cooling to 20-30°C; adding biotin-labeled bases and DNA polymerase to the digested material, and incubating at 35-40°C for 3-5 hours.

[0015] In an embodiment of the present application, the step of intranuclear ligation comprises: adding a DNA ligase-containing ligation reaction system to the end-repaired material, incubating at 15-18°C for 3-5 hours, and incubating at 23-28°C for 0.5-1.5 hours; the DNA ligase-containing ligation reaction system comprises: 250-350 mM Tris-HCl, pH 7.5-8; 80-120 mM magnesium chloride; 80-120 mM dithiothreitol, and 0.5-1.5 mM ATP, 8-12 vol% Triton X-100, and 40-60 U T4 DNA ligase.

[0016] In an embodiment of the present application, the step of DNA decrosslinking comprises: centrifuging the material after intranuclear ligation at 800-1200 rcf for 2-4 minutes at 20-30°C, discarding the supernatant, resuspending with SDS lysis buffer; adding proteinase K, and incubating at 50-60°C for 20-40 minutes; adding salt, and incubating at 60-70°C for 8-12 hours; the SDS lysis buffer comprises: 40-60 mM Tris-HCl, pH 7.5-8.5, 0.5-1.5 vol% SDS, and 8-12 mM ethylenediaminetetraacetic acid.

[0017] In an embodiment of the present application, the step of designing the RNA probe reverse-complementary to the core promoter sequence comprises: synthesizing the RNA probe reverse-complementary to the core promoter sequence according to the plant core promoter library.

[0018] Advantages of the present application:

[0019] The present application provides a method for high-resolution targeted capture of promoter interaction fragments suitable for plants, which specifically captures promoter interaction fragments from a high-resolution chromatin conformation capture library through complementary pairing and hybridization of RNA and DNA, and further obtains information of the promoter interaction fragments. The present application realizes capture through complementary pairing and hybridization of RNA and DNA, does not rely on protein, and can better solve the problem that existing targeted capture technology cannot be used for whole genome capture by immunoprecipitation from the perspective of interaction of DNA element sequences themselves. Compared with existing capture technology, the capture efficiency of the present application is greatly improved, and has obvious advantages in searching for long-distance regulatory elements.

[0020] Of course, implementing any product or method of the present application does not necessarily require achieving all the advantages described above at the same time. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the accompanying drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only represent some of the embodiments of the present application, and other embodiments can also be obtained by those skilled in the art based on these drawings.

[0022] Figure 1 is a flow chart of the promoter interaction fragment capture method of an embodiment of the present application;

[0023] Figure 2A is a comparison of the efficiency of the promoter interaction fragment capture method of the present application and the prior art; Figure 2B is the median length of the promoter loops captured in Example 1; Figure 2C is the median length of the chromatin loops obtained by analysis of Comparative Example 1;

[0024] Figure 3A is the interaction between the intrachromosomal and interchromosomal captured in Example 1; Figure 3B is the interaction of the chromatin loops captured in Example 1 at the promoter region of the probe and the interaction of the promoter and the non-probe region; Figure 3C is the composition of the non-probe region; Figure 3D is the interaction of the intergenic region captured in Example 1 with the proximal gene promoter and the distal gene promoter; Figure 3E is the interaction of the genic region captured in Example 1 with the proximal gene promoter and the distal gene promoter. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments only represent some of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those skilled in the art based on the present application are within the scope of the present application.

[0026] The first aspect of the present application provides a method for high-resolution targeted capture of promoter interaction fragments suitable for plants, comprising: constructing a chromatin conformation capture pre-library; designing an RNA probe reverse complementary to a core promoter sequence; capturing promoter interaction fragments in the chromatin conformation capture pre-library using the RNA probe; library amplification; sequencing to obtain promoter interaction fragment information; wherein the step of capturing promoter interaction fragments in the chromatin conformation capture pre-library using the RNA probe comprises: adding RNase inhibitor and the RNA probe to the chromatin conformation capture pre-library, hybridizing for 16-20 hours to obtain a hybridization mixture; adding streptavidin magnetic beads to the hybridization mixture and mixing, incubating with shaking, centrifuging, resuspending the magnetic beads after discarding the supernatant, incubating for 10-20 minutes, vortexing 1-2 times every 5-10 minutes; centrifuging, resuspending the magnetic beads after discarding the supernatant, incubating at 60-70°C for 8-12 minutes; washing 2-4 times after discarding the supernatant, resuspending the magnetic beads by adding sterile water again.

[0027] In the present application, the centrifugation method of the above step is not particularly limited, as long as the purpose of the present application can be achieved, for example, rapid centrifugation for 4-6 seconds can be used. The method of the above shock incubation is not particularly limited, as long as the purpose of the present application can be achieved, for example, shock incubation at 1000-1500 r / min can be used. The method of the above discarding supernatant is not particularly limited, as long as the purpose of the present application can be achieved, for example, placing in a magnetic stand until the solution is clear, and then aspirating and discarding the supernatant.

[0028] The inventors found in the research that the capture method of the present application can specifically capture the promoter interaction fragments from the high-resolution chromatin conformation capture library. Compared with the existing capture technology, the capture efficiency of the present application is greatly improved, and has obvious advantages in finding long-distance regulatory elements.

[0029] In an embodiment of the present application, the volume ratio of the chromatin conformation capture pre-library, the RNAse inhibitor, and the RNA probe is 3-8:0.3-0.8:1-3; and the volume ratio of the streptavidin magnetic beads and the hybridization mixture is 15:2-4. The inventors found in the research that when the volume ratio of the chromatin conformation capture pre-library, the RNAse inhibitor, and the RNA probe is within the range of the present application, the effectiveness of the RNA probe can be better maintained, the complementarity between the RNA probe and the target region in the library can be better achieved, and the complementation pairing of single-stranded DNA in the library can be avoided to a greater extent. When the volume ratio of the streptavidin magnetic beads and the hybridization mixture is within the range of the present application, the target fragments can be more efficiently enriched.

[0030] In the present application, the temperature generally refers to room temperature, about 20-30°C, unless otherwise specified.

[0031] In an embodiment of the present application, the chromatin conformation capture pre-library further comprises a pretreatment step of concentrating the chromatin conformation capture pre-library using DNA purification magnetic beads. The type of DNA purification magnetic beads is not particularly limited in the present application, as long as the purpose of the present application can be achieved, for example, the DNA purification magnetic beads can be selected from at least one of Qiagen purification magnetic beads (N411-01), Weijinbio purification magnetic beads (DMT-001), and Bemar purification magnetic beads (A63882). Concentrating the chromatin conformation capture pre-library using DNA purification magnetic beads can reduce the volume of the chromatin conformation capture pre-library, so that hybridization capture can be better performed.

[0032] The type of RNAse inhibitor is not particularly limited in the present application, as long as the purpose of the present application can be achieved, for example, the RNAse inhibitor can be selected from at least one of RNAse inhibitor (NF2015) from Diwinbio, RNAse inhibitor (TAG078) from Biaolai, and RNAse inhibitor (2130A) from Junner.

[0033] The application does not make special limitations on the washing method, and any method that can achieve the purpose of the application is acceptable. For example, the washing can be performed by gently blowing the sample with a pipette for 6-10 times after adding the washing buffer. The application does not make special limitations on the mixing method, and any method that can achieve the purpose of the application is acceptable. For example, the mixing can be performed by gently blowing the sample with a pipette for 6-10 times.

[0034] In an embodiment of the application, the step of capturing the promoter interaction fragments in the chromatin conformation capture pre-library using the RNA probe comprises: adding a hybridization buffer, a blocking agent, an RNAse inhibitor and the RNA probe to the chromatin conformation capture pre-library, hybridizing for 16-20 hours to obtain a hybridization mixture; washing the streptavidin magnetic beads, discarding the supernatant, and repeating 2-4 times; resuspending the magnetic beads to obtain a streptavidin magnetic bead resuspension; adding the streptavidin magnetic bead resuspension to the hybridization mixture and mixing, incubating with shaking, centrifuging, resuspending the magnetic beads after discarding the supernatant, and incubating for 10-20 minutes, with vortexing every 5-10 minutes; centrifuging, resuspending the magnetic beads after discarding the supernatant, and incubating at 60-70°C for 8-12 minutes; washing 2-4 times after discarding the supernatant, and resuspending the magnetic beads with sterile water again.

[0035] The application does not make special limitations on the hybridization buffer and the blocking agent, and any buffer and agent that can achieve the purpose of the application are acceptable. For example, the hybridization buffer can be selected from 2x Hyb buffer (kit: Diying Biological, item number NC1003), and the blocking agent can be selected from Blocker Mix (kit: Diying Biological, item number NF2015).

[0036] In an embodiment of the application, the volume ratio of the chromatin conformation capture pre-library, the hybridization buffer, the blocking agent, the RNAse inhibitor and the RNA probe is 3-8: 15-20: 5-10: 0.3-0.8: 1-3, and the volume ratio of the streptavidin magnetic bead resuspension and the hybridization mixture is 15: 2-4. The inventors have found in research that the volume ratio of the chromatin conformation capture pre-library, the hybridization buffer, the blocking agent, the RNAse inhibitor and the RNA probe within the range of the application can better maintain the effectiveness of the RNA probe, better achieve the complementarity of the RNA probe and the target region in the library, and more effectively avoid the complementary pairing of single-stranded DNA in the library. The volume ratio of the streptavidin magnetic bead resuspension and the hybridization mixture within the range of the application can more efficiently enrich the target fragments.

[0037] In an embodiment of the present application, the step of library amplification comprises: performing PCR reaction on the RNA probe-captured promoter interaction fragments; after the PCR reaction is completed, adding DNA purification magnetic beads and incubating for 3-7 minutes; discarding the supernatant after standing for 3-5 minutes, then adding 70-90 vol% ethanol and incubating for 20-40 seconds, discarding the supernatant, then adding 70-90 vol% ethanol and incubating for 20-40 seconds, discarding the supernatant, standing for 1-3 minutes; then adding ultrapure water for elution and standing for 1-3 minutes; taking the supernatant and performing sequencing to obtain the information of the promoter interaction fragments.

[0038] The present application does not make special limitations on the PCR reaction, which can achieve the purpose of the present application, for example, PCR amplification premix solution and primer premix solution can be added in the RNA probe-captured promoter interaction fragments for PCR reaction. The present application does not make special limitations on the PCR amplification premix solution and primer premix solution, which can be directly purchased, for example, the PCR amplification premix solution is purchased from Diwin Biology (item number: NC1003), and the primer premix solution is purchased from Nuoyuan (item number: NDM608).

[0039] In an embodiment of the present application, the volume ratio of the RNA probe-captured promoter interaction fragments, the PCR amplification premix solution and the primer premix solution is 3-5:4-6:1.

[0040] In an embodiment of the present application, the conditions of the PCR reaction are as follows:

[0041] The inventors found in the research that the library amplification method of the present application can better amplify the target fragments, so that the library meets the requirements of sequencing.

[0042] In an embodiment of the present application, the process of the promoter interaction fragment capture method is shown in FIG. 1, which comprises crosslinking, enzyme digestion, biotin labeling, ligation, decrosslinking, ultrasonication, biotin enrichment, library construction, probe hybridization, probe capture, library amplification, and obtaining the information of the promoter interaction fragments.

[0043] In an embodiment of the present application, the step of constructing the plant chromatin conformation capture pre-library comprises: cross-linking the sample to obtain a cross-linked material; performing nuclear extraction on the cross-linked material to obtain a nuclear extraction material; performing restriction enzyme digestion on the nuclear extraction material to obtain a digested material; performing sticky end blunting on the digested material with biotin-labeled bases to obtain a blunted material; performing intranuclear ligation on the blunted material to obtain a ligated material; performing DNA de-cross-linking on the ligated material, extracting DNA, and using ultrasonic to break the DNA fragments, sorting, and performing biotin enrichment with magnetic beads to construct the chromatin conformation capture pre-library.

[0044] In an embodiment of the present application, the step of cross-linking comprises: mixing the sample with a cross-linking system, cross-linking and fixing at 10-30°C for 10-30 minutes, adding glycine to a final concentration of 0.05-0.15M, and treating at 10-30°C for 8-12 minutes to terminate the reaction; and the cross-linking system comprises formaldehyde at a final concentration of 1-3wt%.

[0045] In an embodiment of the present application, the cross-linking system further comprises: 8-12mM KH2PO4(pH 7.0), 40-60mM NaCl, and 0.05-0.2M sucrose.

[0046] The inventors have found in research that the formaldehyde cross-linking method of the present application can better cross-link proteins in cells with DNA, DNA with DNA, preserve their interaction relationship, and maintain the 3D structure in cells.

[0047] In an embodiment of the present application, the step of nuclear extraction comprises: resuspending the cross-linked material with NIB buffer, filtering, collecting the filtrate, centrifuging at 3000-5000rpm at 3-5°C for 8-12 minutes, and discarding the supernatant; resuspending again with NIB buffer, centrifuging at 3000-5000rpm at 3-5°C for 8-12 minutes, discarding the supernatant, and repeating 1-2 times; resuspending again with NIB buffer, centrifuging at 2000-4000rcf at 3-5°C for 3-7 minutes, and discarding the supernatant; and the NIB buffer comprises: 10-30mM HEPES, pH 7.5-8.5; 200-300mM sucrose; 0.5-1.5mM magnesium chloride; 3-8mM potassium chloride; 30-50vol% glycerol; 0.2-0.3vol% Triton X-100; 0.05-0.15mM phenylmethylsulfonyl fluoride; 0.05-0.15vol% protease inhibitor; and 0.05-0.15vol% β-mercaptoethanol.

[0048] In an embodiment of the present application, the mass-volume ratio of the cross-linked material to the NIB buffer is 1:8-12 g / mL.

[0049] The present application does not particularly limit the manner of filtration, and any manner that can achieve the purpose of the present application is acceptable, for example, double-layer nylon membrane, 25-40 μm filter.

[0050] The present application does not particularly limit the type of the protease inhibitor, and any type that can achieve the purpose of the present application is acceptable, for example, the protease inhibitor can be purchased from Thermo (AM2694 or A32955), Abtek (RK21401) or Roche (3335399001).

[0051] The inventors have found in research that the purity and quality of the extracted nuclei are improved by using the nuclear extraction method of the present application.

[0052] In an embodiment of the present application, the step of restriction enzyme digestion comprises: resuspending the nuclear extraction material using 1×RE buffer, centrifuging at 2000-4000 rcf for 3-7 minutes at 3-5°C, and discarding the supernatant; resuspending the precipitate with 0.3-0.7 vol% SDS solution, and incubating at 60-65°C for 3-7 minutes; adding 8-12 vol% Triton X-100, and incubating at 35-40°C for 10-20 minutes; adding 10×RE buffer and restriction enzyme, and incubating at 35-40°C for 12-16 hours; the 1×RE buffer comprises: 0.005-0.015 M sodium chloride; 4-6 mM Tris-HCl, pH 7.5-8.5; 0.8-1.2 mM magnesium chloride; 0.05-0.15 mM dithiothreitol; the 10×RE buffer comprises: 0.05-0.15 M sodium chloride; 40-60 mM Tris-HCl, pH 7.5-8.5; 8-12 mM magnesium chloride; 0.5-1.5 mM dithiothreitol.

[0053] In an embodiment of the present application, the volume ratio of the nuclear extraction material to the 1×RE buffer is 1:2-4.

[0054] In an embodiment of the present application, the ratio of the nuclei to the restriction enzyme is 4-6 million: 50 U; preferably, 5 million nuclei are added with 50 U of the restriction enzyme.

[0055] In the present application, the type of restriction enzyme is not particularly limited, and any restriction enzyme can be used as long as it can achieve the purpose of the present application. In one embodiment of the present application, the restriction enzyme is selected from at least one of DpnII, HindIII and MboI.

[0056] The inventors have found that the restriction enzyme digestion method of the present application can effectively digest DNA into small fragments under chromatin cross-linking conditions.

[0057] In one embodiment of the present application, the step of filling the sticky ends comprises incubating the digested material at 60-65°C for 15-25 minutes, and then cooling to 20-30°C; adding biotin-labeled bases and DNA polymerase to the digested material, and incubating at 35-40°C for 3-5 hours.

[0058] In one embodiment of the present application, the DNA polymerase is selected from Klenow polymerase. In the present application, the type of Klenow polymerase is not particularly limited, and any Klenow polymerase can be used as long as it can achieve the purpose of the present application. For example, the Klenow polymerase can be purchased from NEB (M0210V), Thermo (18012096) or Novagen (N104-01).

[0059] The inventors have found that the sticky end filling method of the present application can better fill the sticky ends cut by the restriction enzyme into blunt ends, thereby marking the ligation site.

[0060] In one embodiment of the present application, the step of in-nucleus ligation comprises adding a DNA ligase-containing ligation reaction system to the end-filled material, incubating at 15-18°C for 3-5 hours, and incubating at 23-28°C for 0.5-1.5 hours; the DNA ligase-containing ligation reaction system comprises 250-350 mM Tris-HCl, pH 7.5-8; 80-120 mM magnesium chloride; 80-120 mM dithiothreitol and 0.5-1.5 mM ATP, 8-12 vol% Triton X-100 and 40-60 U T4 DNA ligase.

[0061] The inventors have found that the in-nucleus ligation method of the present application can better ligate DNA blunt-end small fragments that are close in space and have been filled in the nucleus.

[0062] In an embodiment of the present application, the step of DNA de-crosslinking comprises: centrifuging the post-connection material in the nucleus at 800-1200 rcf for 2-4 minutes at 20-30 °C, discarding the supernatant, resuspending with SDS lysis buffer; adding proteinase K, incubating at 50-60 °C for 20-40 minutes; adding salt, incubating at 60-70 °C for 8-12 hours; the SDS lysis buffer comprises: 40-60 mM Tris-HCl, pH 7.5-8.5, 0.5-1.5 vol% SDS, 8-12 mM ethylenediaminetetraacetic acid.

[0063] The kind of the proteinase K is not particularly limited in the present application as long as the purpose of the present application can be achieved, for example, the proteinase K can be purchased from Merck (1245680100), Thermo (M0055) or NEB (P8107S); the kind of the salt is not particularly limited in the present application as long as the purpose of the present application can be achieved, for example, the salt can be selected from at least one of NaCl, KCl and MgCl2.

[0064] The inventors found in the research that the DNA de-crosslinking method of the present application can better digest the proteins in the nucleus, dissociate and degrade the proteins from the DNA.

[0065] In an embodiment of the present application, the step of extracting DNA comprises: after DNA de-crosslinking, adding a phenol: chloroform: isopropyl alcohol solution with a volume ratio of 24-26: 23-25: 1, centrifuging at 8000-12000 rcf for 4-6 minutes, collecting the upper solution, adding sodium acetate buffer and isopropyl alcohol, and incubating at -15 to -25 °C for 20-40 minutes; centrifuging at 10000-15000 rcf for 15-25 minutes at 3-5 °C, washing the precipitate with pre-cooled 75-85 vol% ethanol, discarding the supernatant, and dissolving with TE buffer; adding ribonuclease A (RNase A), and incubating at 35-40 °C for 20-40 minutes; according to the total volume of DNA, adding 1 / 10 volume of sodium acetate buffer and equal volume of isopropyl alcohol, centrifuging at 10000-15000 rcf for 15-25 minutes at 3-5 °C, washing the precipitate with pre-cooled 75-85 vol% ethanol, discarding the supernatant, and dissolving the DNA with Tris elution buffer to obtain purified DNA; wherein the sodium acetate buffer comprises 2-4 M sodium acetate, pH 5-5.5; the TE buffer comprises 8-12 mM Tris-HCl, pH 7.5-8.5, 0.5-1.5 mM EDTA; and the Tris elution buffer comprises 8-12 mM Tris-HCl, pH 7.5-8.5.

[0066] In an embodiment of the present application, the ultrasonic disruption of the DNA fragments is 200-600 bp. The method of ultrasonic disruption is not particularly limited in the present application, as long as it can achieve the purpose of the present application.

[0067] The inventors have found in research that the method for extracting DNA of the present application can better extract DNA from a solution, meeting the requirements of subsequent ultrasonic disruption.

[0068] In an embodiment of the present application, the sorting step comprises: adding DNA purification magnetic beads to the ultrasonic disruption of the DNA fragments, and incubating for 4-6 minutes; clarifying on a magnetic stand, and centrifuging at 8000-12000rcf for 0.5-1.5 minutes; clarifying on a magnetic stand, and taking the supernatant; adding 1 / 4 volume of DNA purification magnetic beads to the supernatant, and incubating for 4-6 minutes; clarifying on a magnetic stand, and discarding the supernatant; adding 75-85vol% ethanol, and incubating for 0.5-1.5 minutes; adding 75-85vol% ethanol again, and incubating for 0.5-1.5 minutes; discarding the supernatant, and drying the magnetic beads until no ethanol remains; resuspending the magnetic beads in Tris elution buffer, and incubating for 4-6 minutes; taking the supernatant, and obtaining the sorted DNA; wherein the Tris elution buffer comprises 8-12mM Tris-HCl, pH 7.5-8.5.

[0069] The inventors have found in research that the sorting method of the present application can more efficiently and accurately obtain DNA fragments of a desired size, meeting the requirements of sequencing length.

[0070] In an embodiment of the present application, the biotin enrichment step comprises: resuspending C1 magnetic beads in TWB buffer, clarifying on a magnetic stand, and discarding the supernatant; resuspending the magnetic beads in BB buffer; adding the C1 magnetic beads to the sorted DNA, and incubating for 10-20 minutes; clarifying on a magnetic stand, and discarding the supernatant; resuspending the magnetic beads in TWB buffer, incubating for 1-3 minutes, clarifying on a magnetic stand, discarding the supernatant, resuspending the magnetic beads in TWB buffer, incubating for 1-3 minutes, clarifying on a magnetic stand, discarding the supernatant, and resuspending the magnetic beads in Tris elution buffer; clarifying on a magnetic stand; discarding the supernatant, and resuspending the magnetic beads in Tris elution buffer; wherein the TWB buffer comprises: 4-6mM Tris-HCl, pH 7.5-8.5; 0.4-0.6mM EDTA; 0.8-1.2M NaCl; 0.04-0.06vol% Tween-20; and the BB buffer comprises: 8-12mM Tris-HCl, pH 7.5-8.5; 0.8-1.2mM EDTA, 1-3M NaCl.

[0071] The inventors found in the research that the biotin-enriched method of the application can better enrich the DNA fragments with biotin labeling.

[0072] The method for constructing the chromatin conformation capture pre-library is not particularly limited in the application, and any method that can achieve the purpose of the application can be used, for example, a commercial DNA library kit can be used to construct the chromatin conformation capture pre-library according to the instructions.

[0073] In an embodiment of the application, the step of designing the RNA probe reverse complementary to the core promoter sequence comprises: synthesizing the RNA probe reverse complementary to the core promoter sequence according to the plant core promoter library; preferably, when the plant is Arabidopsis thaliana, the RNA probe sequence is as shown in SEQ ID NO: 1 to SEQ ID NO: 34498.

[0074] The method for capturing the promoter interaction fragment of the application can specifically capture the promoter interaction fragment from the high-resolution chromatin conformation capture library through the complementary pairing and hybridization of RNA and DNA, and then obtain the information of the promoter interaction fragment. Compared with the existing capture technology, the capture efficiency of the application is greatly improved, and the application has obvious advantages in finding long-distance regulatory elements.

[0075] EMBODIMENT

[0076] Hereinafter, the embodiments of the application will be described more specifically by citing examples and comparative examples. Various tests and evaluations were performed according to the following methods. In addition, unless otherwise specified, "parts", "%", are mass-based, "room temperature" is 20-30°C, and "overnight" is 12-16 hours. The experimental materials and methods used, unless otherwise specified, are conventional materials and methods.

[0077] EMBODIMENT 1

[0078] (1) Plant tissue cross-linking

[0079] The Arabidopsis seedlings (Col-0) were treated with darkness at 4°C for 2 days, and then grown on 1 / 2MS medium for 10 days under the conditions of 22°C, 16h light / 8h darkness. 1.5g of the Arabidopsis seedlings grown for 10 days were collected and placed in 30mL MC solution (10mM KH2PO4(PH 7.0), 50mM NaCl, 0.1M sucrose, prepared with ddH2O), and formaldehyde was added to a final concentration of 2wt%, and cross-linking and fixation were performed at room temperature for 20 minutes. Then, glycine was added to a final concentration of 0.1M, and the reaction was terminated by treatment at room temperature for 10 minutes.

[0080] (2) Nuclei extraction

[0081] The 3 g of crosslinked material was ground into powder with liquid nitrogen, resuspended with 30 mL of NIB buffer, filtered with double-layer nylon membrane, and the filtrate was collected in a 50 mL centrifuge tube. The sample was centrifuged at 4000 rpm for 10 minutes at 4°C, and the supernatant was discarded. The sample was resuspended with 10 mL of NIB buffer, centrifuged at 4000 rpm for 10 minutes at 4°C, and the supernatant was discarded. The sample was resuspended with 10 mL of NIB buffer, centrifuged at 4000 rpm for 10 minutes at 4°C, and the supernatant was discarded. The sample was resuspended with 1 mL of NIB buffer and transferred to a 1.5 mL enzyme-free centrifuge tube. The sample was centrifuged at 3000 rcf for 5 minutes at 4°C, and the supernatant was discarded to obtain the nuclear extract material. The concentration of the nuclear extract material was estimated using a cell counter under a fluorescence microscope, and 150-200 million nuclei were used for subsequent experiments.

[0082] The NIB buffer was 20 mM HEPES, pH 8.0; 250 mM sucrose; 1 mM magnesium chloride; 5 mM potassium chloride; 40 vol% glycerol; 0.25 vol% Triton X-100; 0.1 mM benzylsulfonyl fluoride; 0.1 vol% protease inhibitor cocktail (purchased from Sigma, catalog number A32955); and 0.1 vol% β-mercaptoethanol.

[0083] (3) Restriction enzyme digestion

[0084] The prepared nuclear extract material was gently resuspended with 300 μL of 1× RE buffer, centrifuged at 3000 rcf for 5 minutes at 4°C, and the supernatant was discarded. The precipitate was resuspended with 0.5 vol% SDS solution and adjusted to 150 μL, and an equal amount of the resuspension was transferred to three 1.5 mL enzyme-free centrifuge tubes. The samples were incubated at 62°C for 5 minutes. In each centrifuge tube, 145 μL of deionized water and 25 μL of 10 vol% Triton X-100 were added, the centrifuge tube was gently inverted to suspend the precipitate in the liquid, and the sample was incubated at 37°C for 15 minutes. In each centrifuge tube, 25 μL of 10× RE buffer and 50 U of restriction enzyme DpnII were added, and the sample was gently mixed and incubated at 37°C overnight to obtain the digested material. The 1× RE buffer was 0.01 M sodium chloride; 5 mM Tris-HCl, pH 7.9; 1 mM magnesium chloride; and 0.1 mM DTT. The 10× RE buffer was 0.1 M NaCl; 50 mM Tris-HCl, pH 7.9; 10 mM MgCl2; and 1 mM DTT.

[0085] (4) Sticky end filling

[0086] The above-prepared enzyme-digested material was incubated at 62°C for 20 minutes, and then cooled to room temperature; 1 μL of 10 mM dTTP, 1 μL of 10 mM dATP, 1 μL of 10 mM dGTP, 25 μL of 0.4 mM biotin-14-dCTP, 14 μL of deionized water, and 40 U of DNA polymerase (Klenow fragment, purchased from NEB, catalog number M0210V) were added to each centrifuge tube, mixed gently, and incubated at 37°C for 4 hours to obtain the end-repaired material.

[0087] (5) Intranuclear ligation

[0088] To each centrifuge tube containing the above-prepared end-repaired material, 663 μL of deionized water, 120 μL of blunt-end ligation buffer, 100 μL of 10 vol% Triton X-100, and 50 U of T4 DNA ligase were added, mixed gently, and then incubated at 16°C for 4 hours and at 25°C for 1 hour to obtain the intranuclear ligation material. The blunt-end ligation buffer was 300 mM Tris-HCl, pH 7.8; 100 mM magnesium chloride; 100 mM DTT; and 1 mM ATP.

[0089] (6) DNA de-crosslinking

[0090] The above-prepared intranuclear ligation material was centrifuged at 1000 rcf for 3 minutes at room temperature, the supernatant was discarded, and the material was resuspended with 750 μL of SDS lysis buffer; 10 μL of proteinase K (18 mg / mL, purchased from Merck, catalog number 1245680100) was added to each centrifuge tube, and the mixture was incubated at 55°C for 30 minutes; 30 μL of 5 M sodium chloride solution was added to each centrifuge tube, and the mixture was incubated at 65°C overnight. The SDS lysis buffer was 50 mM Tris-HCl, pH 8.0; 1 vol% SDS; and 10 mM EDTA.

[0091] (7) DNA extraction

[0092] In each centrifugal tube, 750 μL of a phenol, chloroform and isoamyl alcohol mixed solution (volume ratio of phenol: chloroform: isoamyl alcohol is 25:24:1) was added, vortexed to mix thoroughly, and then centrifuged at 10000 rcf for 5 minutes. The upper solution was carefully transferred to a new 2 mL enzyme-free centrifugal tube. 75 μL of sodium acetate buffer and 750 μL of isopropanol were added to each centrifugal tube, and repeated inversion was performed until complete mixing. Incubation was performed at -20°C for 30 minutes. Centrifugation was performed at 13000 rcf at 4°C for 20 minutes. The precipitate was washed with 1 mL of pre-cooled 80 vol% ethanol, and after the supernatant was discarded, the centrifugal tube was placed on the clean bench until there was no obvious liquid on the surface of the precipitate. 100 μL of TE buffer was used to dissolve the DNA. The total volume of DNA was measured using a pipette, and 1 / 10 volume of sodium acetate buffer and an equal volume of isopropanol were added. Inversion was performed until complete mixing. Centrifugation was performed at 13000 rcf at 4°C for 20 minutes. The precipitate was washed with 1 mL of pre-cooled 80 vol% ethanol, and after the supernatant was discarded, the centrifugal tube was placed on the clean bench until there was no obvious liquid on the surface of the precipitate. 100 μL of Tris elution buffer was used to dissolve the DNA. The DNA concentration was measured using qubit fluorescence quantification, the total mass of DNA was calculated, and 2 μL of DNA was used for agarose gel electrophoresis to detect the ligation efficiency, and to ensure that the total amount of DNA was more than 1.6 μg for subsequent experiments. The sodium acetate buffer is 3M sodium acetate, pH 5.2. The TE buffer is 10 mM Tris-HCl, pH 8.0; 1 mM EDTA. The Tris elution buffer is 10 mM Tris-HCl, pH 8.0.

[0093] (8) Ultrasonic treatment

[0094] The DNA was transferred to an enzyme-free PCR tube at 100 μL, and the DNA was broken to 200-600 bp using an ultrasonic instrument. The fragment size was detected using agarose gel electrophoresis during the process.

[0095] (9) Sorting

[0096] Elute the DNA in the PCR tube to 100 μL with Tris elution buffer; add 60 μL of equilibrated DNA purification magnetic beads (purchased from Novagen, product number N411-01), mix well by repeatedly aspirating with a pipette gun, and incubate at room temperature for 5 minutes; place the PCR tube on a magnetic stand for 5 minutes; after centrifugation at 10,000 rcf for 1 minute at room temperature, place the PCR tube back on the magnetic stand until the solution is clear; transfer the supernatant to a new enzyme-free PCR tube, and discard the magnetic beads; add 1 / 4 volume of DNA purification magnetic beads to the supernatant, mix well by repeatedly aspirating with a pipette gun, and incubate at room temperature for 5 minutes; place the PCR tube on a magnetic stand for 5 minutes until the solution is clear; discard the supernatant, and keep the PCR tube on the magnetic stand, add 200 μL of 80 vol% ethanol to the PCR tube, and incubate at room temperature for 1 minute; discard the supernatant, and keep the PCR tube on the magnetic stand, add 200 μL of 80 vol% ethanol to the PCR tube, and incubate at room temperature for 1 minute; discard the supernatant, and keep the PCR tube on the magnetic stand, open the cap, and air-dry the magnetic beads until no ethanol remains; remove the PCR tube from the magnetic stand, resuspend the magnetic beads in 50 μL of Tris elution buffer, and incubate at room temperature for 5 minutes; place the PCR tube on a magnetic stand, incubate until the solution is clear, and transfer the supernatant to a new enzyme-free PCR tube; measure the total mass of DNA using a Qubit, and detect the size of the sorted DNA concentrated at 200-500 bp using agarose gel electrophoresis. Tris elution buffer: 10 mM Tris-HCl, pH 8.0.

[0097] (10) Biotin enrichment

[0098] Resuspend 10 μL of C1 magnetic beads (purchased from Invitrogen, product number 01332376) in 1.5 mL enzyme-free centrifuge tubes with 300 μL of filtered TWB buffer; place the centrifuge tube on a magnetic stand for 2 minutes until the solution is clear, discard the supernatant, and resuspend the magnetic beads with 50 μL of filtered BB buffer; wherein the TWB buffer: 5 mM Tris-HCl, pH 8.0; 0.5 mM EDTA, 1 M NaCl, 0.05 vol% Tween-20. The BB buffer: 10 mM Tris-HCl, pH 8.0; 1 mM EDTA, 2 M NaCl.

[0099] The DNA obtained from step (9) is diluted to 50 μL using Tris elution buffer, 50 μL of the above-mentioned BB buffer is added to resuspend the C1 magnetic beads, and the mixture is mixed well using a pipette gun and incubated at room temperature for 15 minutes; the centrifuge tube is placed on a magnetic stand until the solution is clear; the supernatant is discarded, the centrifuge tube is removed from the magnetic stand, the magnetic beads are resuspended with 200 μL of TWB buffer, and incubated at room temperature for 2 minutes before being placed on a magnetic stand until the solution is clear; the supernatant is discarded, the centrifuge tube is removed from the magnetic stand, the magnetic beads are resuspended with 200 μL of TWB buffer, and incubated at room temperature for 2 minutes before being placed on a magnetic stand until the solution is clear; the supernatant is discarded, the centrifuge tube is removed from the magnetic stand; the magnetic beads are resuspended with 100 μL of Tris elution buffer, and then the centrifuge tube is placed on a magnetic stand until the solution is completely clear; the supernatant is discarded, the centrifuge tube is removed from the magnetic stand; the magnetic beads are resuspended with 48 μL of Tris elution buffer to obtain biotin-enriched DNA.

[0100] (11) Chromatin conformation capture pre-library construction

[0101] Library construction is performed using the VAHTS Universal Pro DNA Library Prep Kit for MGI NDM608-01 kit (purchased from Novogene, item number NDM608), and the following reagents are from the VAHTS Universal Pro DNA Library Prep Kit for MGI NDM608-01 kit unless otherwise specified.

[0102] End repair: After thawing the end repair buffer (End Prep Buffer), mix well by inverting, and prepare the following reaction in a sterile PCR tube:

[0103] Mix gently by pipetting (do not mix by vortexing), and place the PCR tube in a PCR instrument to perform the following reaction:

[0104] Linker ligation: Prepare the following reaction in the PCR tube from the previous step:

[0105] Mix gently by pipetting (do not mix by vortexing), and place the PCR tube in a PCR instrument to perform the following reaction:

[0106] Place the centrifuge tube on the magnetic stand until the solution is completely clear, discard the supernatant, and remove the centrifuge tube from the magnetic stand; resuspend the magnetic beads with 150 μL of TWB buffer, incubate at room temperature for 2 minutes, then place the centrifuge tube on the magnetic stand until the solution is completely clear, discard the supernatant, and remove the centrifuge tube from the magnetic stand; resuspend the magnetic beads with 150 μL of TWB buffer, incubate at room temperature for 2 minutes, then place the centrifuge tube on the magnetic stand until the solution is completely clear, discard the supernatant, and remove the centrifuge tube from the magnetic stand; resuspend the magnetic beads with 100 μL of Tris elution buffer, place the centrifuge tube on the magnetic stand until the solution is completely clear, discard the supernatant, and remove the centrifuge tube from the magnetic stand; resuspend the magnetic beads with 21 μL of Tris elution buffer; place the centrifuge tube in a 98°C incubator for 10 minutes, and immediately proceed to the next step after completion; place the centrifuge tube on the magnetic stand until the solution is completely clear, and carefully transfer 20 μL of the supernatant to a new enzyme-free PCR tube to obtain the purified or sorted ligation linker product;

[0107] Amplification reaction: After thawing the PCR primer pre-mix (PCR Primer Mix 3 for lllumina) and the amplification pre-mix (VAHTS HiFi Amplification Mix), mix well by inverting, and prepare the following reaction in a sterile PCR tube:

[0108] Mix gently by pipetting (do not mix by vortexing), and place the PCR tube in a PCR instrument to perform the following reaction:

[0109] After the PCR is completed, add 40 μL of DNA purification magnetic beads (purchased from Novozyme, product number N411-01) to the tube, mix well by pipetting 8-10 times, and incubate at room temperature for 5 minutes; place the tube on a magnetic stand for 3-5 minutes until the solution is clear, and then aspirate the supernatant; do not remove the centrifuge tube from the magnetic stand, add 150 μL of 80 vol% ethanol, incubate at room temperature for 30 seconds, and discard the supernatant; add another 150 μL of 80 vol% ethanol, incubate at room temperature for 30 seconds, and discard the supernatant; let stand at room temperature for 1-3 minutes until there is no obvious liquid residue on the surface of the magnetic beads, which is just not shiny or matte; remove the tube from the magnetic stand, add 20 μL of ultrapure water for elution, mix well by pipetting 8-10 times, and let stand at room temperature for 2 minutes; place the tube on a magnetic stand, aspirate the supernatant after the solution is clear, and transfer it to a new enzyme-free PCR tube. Measure the total DNA mass using Qubit, and aspirate no less than 1 μg of DNA pre-library to store at -20°C; the rest of the pre-library is subjected to second-generation sequencing, and after ensuring the quality of the library, targeted capture is performed.

[0110] (12) RNA probe design

[0111] According to published data, the 165bp upstream to 5bp downstream of the gene transcription initiation site of plants includes the main promoter cis-element, so we choose the above interval of all genes in plants as the plant core promoter library (Jores T, Tonnies J, Wrightsman T, Buckler ES, Cuperus JT, Fields S, Queitsch C: Synthetic promoter designs enabled by a comprehensive analysis of plant core promoters. Nat Plants 2021, 7:842-855.), and the RNA probe complementary to the core promoter sequence is synthesized (synthesized by Diwin Biotechnology), and the RNA probe sequence is shown as SEQ ID NO: 1 to SEQ ID NO: 34498.

[0112] (13) Probe capture

[0113] Probe capture was performed using QuarHyb One Reagent Kit (purchased from Diwin Biotechnology, item number NF2015), and the following reagents were from QuarHyb One Reagent Kit unless otherwise specified;

[0114] The pre-library constructed in step (11) was diluted to 50 μL in an enzyme-free PCR tube, and the library was concentrated to 5 μL using DNA purification magnetic beads (purchased from Novozyme, item number N411-01) ; 18 μL of 2xHyb buffer (kit: Diwin Biotechnology item number NC1003, 65°C preheating), 7.5 μL of Blocker Mix (kit: Diwin Biotechnology item number NF2015), 0.5 μL of RNase inhibitor (Diwin Biotechnology, NF2015) and 2 μL of RNA probe were added to the PCR tube, and the mixture was mixed by blowing and sucking with a pipette gun, and was placed in a PCR instrument for the following reaction; hybridization for 16-20 hours to obtain a hybridization mixture;

[0115] 5 μL of equilibrated streptavidin magnetic beads (purchased from Diwin Biotechnology, item number NC1003) was taken into a new enzyme-free PCR tube, and Wash Buffer I was added, and the magnetic beads were mixed by gently blowing with a pipette gun for 8 times; the PCR tube was placed on a magnetic stand, and the supernatant was discarded after clarification; Wash Buffer I was added again, and the magnetic beads were mixed by gently blowing with a pipette gun for 8 times; Wash Buffer I was added again, and the magnetic beads were mixed by gently blowing with a pipette gun for 8 times; the supernatant was removed, and only the magnetic beads were left in the tube bottom; finally, 150 μL of Wash Buffer I was added to each well to resuspend the magnetic beads, and the mixture was placed at room temperature for use;

[0116] After hybridization, make sure the volume of the hybridization system is not less than 20 μL, open the PCR tube on the PCR instrument, add the above-mentioned 150 μL magnetic bead resuspension solution to the hybridization mixture, and use a pipette to gently blow and beat 8 times, and transfer to a new enzyme-free 1.5 mL centrifuge tube; shake in a metal bath at 25°C at 1000-1500 r / min for room temperature shaking incubation; centrifuge quickly for 5 seconds, and place on a magnetic stand for 2-5 minutes to ensure that the liquid is clear, and the supernatant is aspirated; remove the centrifuge tube from the magnetic stand, add 150 μL of wash buffer II (Wash Buffer 2) to resuspend the magnetic beads, and use a pipette to gently blow and beat 10 times, and incubate at room temperature for 15 minutes, vortex once every 5 minutes; centrifuge quickly for 5 seconds, and transfer the liquid to a new enzyme-free PCR tube, place on a magnetic stand, and after the liquid is clear, aspirate the supernatant, and place on a hybridization PCR instrument at 65°C; immediately add 150 μL of 65°C preheated wash buffer III (Wash Buffer 3) to the mixture, and mix the magnetic beads thoroughly on the PCR instrument by blowing and sucking 10 times, and incubate at 65°C for 10 minutes; immediately remove from the PCR instrument and place on a magnetic stand, and after the liquid is clear, aspirate the supernatant as soon as possible, and finally add 20 μL of sterile water to resuspend the magnetic beads.

[0117] (14) Library amplification

[0118] Add 25 μL of PCR amplification premix solution (PCR Mix, purchased from Diwin, item number: NC1003) and 5 μL of primer premix solution (Post-Primer Mix, purchased from Novozyme, item number: NDM608) to the PCR tube, mix well using a pipette, and place in a PCR instrument to perform the following reaction:

[0119] After PCR is completed, add 40 μL of DNA purification magnetic beads (purchased from Novozyme, item number: N411-01) to the mixture, mix thoroughly by blowing and beating 8-10 times with a pipette, and incubate at room temperature for 5 minutes; place on a magnetic stand for 3-5 minutes, aspirate the supernatant after the solution is clear; keep the centrifuge tube on the magnetic stand without removing it, add 150 μL of 80 vol% ethanol, incubate at room temperature for 30 seconds, and discard the supernatant; add another 150 μL of 80 vol% ethanol, incubate at room temperature for 30 seconds, and discard the supernatant; stand at room temperature for 1-3 minutes until there is no obvious liquid residue on the surface of the magnetic beads, just no gloss or matte appearance; remove the tube from the magnetic stand, add 20 μL of ultrapure water for elution, mix well by blowing and beating 8-10 times with a pipette, and stand at room temperature for 2 minutes; place on a magnetic stand, aspirate the supernatant after the solution is clear, transfer to a new enzyme-free PCR tube, and store in a -20°C refrigerator, and wait for library inspection and machine operation.

[0120] Comparative Example 1

[0121] Steps (12) to (14) were not performed, and steps (1) to (11) were the same as in Example 1.

[0122] Effective read statistics method

[0123] The raw sequencing data of Promoter Capture Hi-C (PCHi-C) in the present application is first processed by fastp software for preliminary processing, and low-quality reads and adapter sequences are removed. Then bowtie2 is called by HiC-Pro to align the clean reads back to the Arabidopsis thaliana reference genome. In order to obtain effective interaction fragments, the present application uses HiCUP software to obtain effective chromatin interactions from the original sequence alignment results and remove repeated interactions. The promoter-related interactions obtained by the PCHi-C method in the whole genome range cannot be directly corrected for bias and standardized by traditional Hi-C methods in the data analysis step due to the characteristics of uneven capture efficiency, high requirements for robustness in identifying significant interactions, etc. Therefore, the present application uses the CHICAGO software developed for PCHi-C to identify effective promoter-related interactions, remove experimental-induced bias, and obtain significant promoter-related chromatin loops. The present application adjusts the weight alpha (weightAlpha), weight beta (weightBeta), weight gamma (weightGamma), and weight increment (weightDelta) parameters in CHICAGO to 13.5319239, -1.3100426, -10.3516115, and 0.1635212 for Arabidopsis thaliana genome to eliminate the bias caused by distance effect.

[0124] Effect analysis

[0125] High-resolution chromosome conformation capture technology can capture all interactions in the genome simultaneously in principle, however, the information of high-resolution chromosome conformation capture library is very complex. Therefore, it is difficult to achieve the required sequencing depth to obtain promoter-targeted fragments. In the published high-resolution chromosome conformation capture library of Arabidopsis thaliana, promoter-related reads accounted for only 4% of the entire library (see Yin, X., Romero-Campero, F. J., Yang, M., Baile, F., Cao, Y., Shu, J., Luo, L., Wang, D., Sun, S., Yan, P., et al. (2023). Binding by the Polycomb complex component BMI1 and H2A monoubiquitination shape local and long-range interactions in the Arabidopsis genome. Plant Cell 35: 2484-2503. 10.1093 / plcell / koad112.). In contrast, the capture efficiency can be increased by nearly 10 times using the capture technology of Example 1 of the present application (Figure 2A, where "Data 1 of the present application" is the first repetition of Example 1, and "Data 2 of the present application" is the second repetition of Example 1). The median length of the promoter loops captured in Example 1 of the present application is about 27 kb, and there is no obvious difference between different chromosomes (Figure 2B, where "Chr1" to "Chr5" represent chromosomes 1 to 5, respectively), while the median length of the chromatin loops analyzed by the conformation capture technology of Comparative Example 1 is about 4.5 kb (Figure 2C, where "Chr1" to "Chr5" represent chromosomes 1 to 5, respectively), which further verifies the advantage of the capture technology of the present application in searching for long-distance regulatory elements.

[0126] In addition, it is known that enhancers, silencers and other DNA regulatory elements play a very important role in gene transcription regulation. However, in plants, due to the lack of specific histone modification markers and technical limitations, the search and identification of these regulatory elements is very limited. The present application first develops this method of high-resolution targeted capture of promoter interaction fragments, and successfully completes the experiment in the model plant Arabidopsis thaliana. Promoter-related reads are obtained using the effective read statistics method described above, and the effective reads are visualized using Juicebox and pyGenomeTracks software, and the results are as follows:

[0127] In the embodiment 1 of the present application, 24839 promoter-associated chromatin loops were identified, almost all of which were intrachromosomal interactions rather than interchromosomal interactions (Figure 3A, wherein "Chr1" to "Chr5" represent chromosome 1 to chromosome 5, respectively). In addition, about 75% of the chromatin loops were interactions between two promoters in the probe region, and the remaining 25% of the chromatin loops were interactions between a promoter and a non-probe region (Figure 3B, wherein "Chr1" to "Chr5" represent chromosome 1 to chromosome 5, respectively), which contained other promoters, intergenic and genic regions. In summary, 20460 promoter-promoter chromatin loops, 1194 promoter-intergenic chromatin loops and 3185 promoter-genic chromatin loops were found in the present application (Figure 3C), which indicated that promoters in Arabidopsis tend to interact with other promoters. Moreover, it was found that the intergenic and genic regions not only interact with the adjacent gene promoters, but also interact with the distant gene promoters (Figures 3D to 3E), which is similar to the long-range regulatory elements in mammals. Therefore, the element enrichment of the intergenic and genic regions was analyzed (as shown in Tables 1 to 2), and the cis-elements that can be bound by E2F / DP or MYB ranked first in the motif enrichment analysis of the intergenic or genic regions, which indicated that these transcription factors can be involved in the regulation of non-promoter chromatin loops. In summary, it was found in the present application that promoters in Arabidopsis tend to interact with other promoters; and the intergenic or genic regions interacting with the promoters can be potential enhancers or long-range regulatory elements.

[0128] Table 1

[0129] Table 2

[0130] In the present application, the promoter interaction fragments were specifically captured from the high-resolution chromatin conformation capture library through the complementary pairing and hybridization of RNA and DNA, and then the information of the promoter interaction fragments was obtained. Compared with the existing capture technology, the capture efficiency of the present application is greatly improved, and has obvious advantages in finding long-range regulatory elements.

[0131] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the scope of protection of the present application.

Claims

1. A method for high-resolution targeted capture of promoter interacting fragments suitable for plants, comprising: constructing a chromatin conformation capture pre-library; designing an RNA probe reverse complementary to a core promoter sequence; capturing promoter interacting fragments in the chromatin conformation capture pre-library using the RNA probe; library amplification;sequencing to obtain promoter interacting fragment information; wherein the step of capturing promoter interacting fragments in the chromatin conformation capture pre-library using the RNA probe comprises: adding RNase inhibitor and the RNA probe to the chromatin conformation capture pre-library, hybridizing for 16-20 hours to obtain a hybridization mixture;adding streptavidin magnetic beads to the hybridization mixture, mixing, shaking and incubating, centrifuging, resuspending the magnetic beads after discarding the supernatant, incubating for 10-20 minutes, vortexing 1-2 times every 5-10 minutes, centrifuging, resuspending the magnetic beads after discarding the supernatant, incubating at 60-70℃ for 8-12 minutes;washing 2-4 times after discarding the supernatant, resuspending the magnetic beads again by adding sterile water.

2. The method of claim 1, wherein, The volume ratio of the chromatin conformation capture pre-library, the RNase inhibitor and the RNA probe is 3-8: 0.3-0.8: 1-3;The volume ratio of the streptavidin magnetic beads to the hybridization mixture is 15: 2-4.

3. The method of claim 1, wherein, The step of library amplification comprises: performing PCR reaction on the promoter interacting fragments captured by the RNA probe;after the PCR reaction is completed, adding DNA purification magnetic beads and incubating for 3-7 minutes;standing for 3-5 minutes, then discarding the supernatant, adding 70-90 vol% ethanol and incubating for 20-40 seconds, adding 70-90 vol% ethanol again, incubating for 20-40 seconds, discarding the supernatant, standing for 1-3 minutes, adding ultrapure water to elute, and standing for 1-3 minutes;taking the supernatant and sequencing to obtain promoter interacting fragment information.

4. The method of claim 1, wherein, The step of constructing a plant chromatin conformation capture pre-library comprises: cross-linking the sample to obtain cross-linked material;extracting the nuclei from the cross-linked material to obtain nuclear extract material;performing restriction enzyme digestion on the nuclear extract material to obtain enzyme-digested material;performing sticky end smoothing on the enzyme-digested material using biotin-labeled bases to obtain end-smoothed material;performing intranuclear ligation on the end-smoothed material to obtain intranuclear ligation material;performing DNA de-cross-linking, extracting DNA, and using ultrasonic waves to break the DNA fragments, sorting, and performing biotin enrichment using magnetic beads to construct a chromatin conformation capture pre-library.

5. The method of claim 4, wherein, The step of extracting the nuclei comprises: resuspending the cross-linked material using NIB buffer, filtering, collecting the filtrate, centrifuging at 3000-5000 rpm for 8-12 minutes at 3-5℃, and discarding the supernatant;resuspending again using NIB buffer, centrifuging at 3000-5000 rpm for 8-12 minutes at 3-5℃, discarding the supernatant, and repeating 1-2 times;resuspending again using NIB buffer, centrifuging at 2000-4000 rcf for 3-7 minutes at 3-5℃, and discarding the supernatant. The NIB buffer comprises: 10-30 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH 7.5-8.5; 200-300 mM sucrose; 0.5-1.5 mM magnesium chloride; 3-8 mM potassium chloride; 30-50 vol% glycerol; 0.2-0.3 vol% Triton X-100; 0.05-0.15 mM benzylsulfonyl fluoride; 0.05-0.15 vol% protease inhibitor; 0.05-0.15 vol% β-mercaptoethanol.

6. The method of claim 4, wherein, The step of restriction enzyme digestion comprises: The nuclear extract material is resuspended using 1×RE buffer, centrifuged at 2000-4000 rcf for 3-7 minutes at 3-5 ℃, and the supernatant is discarded; the precipitate is resuspended with 0.3-0.7 vol% SDS solution, and incubated at 60-65 ℃ for 3-7 minutes; 8-12 vol% Triton X-100 is added, and incubated at 35-40 ℃ for 10-20 minutes; 10×RE buffer and restriction enzyme are added, and incubated at 35-40 ℃ for 12-16 hours; The 1×RE buffer comprises: 0.005-0.015 M sodium chloride; 4-6 mM Tris-HCl, pH 7.5-8.5; 0.8-1.2 mM magnesium chloride; 0.05-0.15 mM dithiothreitol; and the 10×RE buffer comprises: 0.05-0.15 M sodium chloride; 40-60 mM Tris-HCl, pH 7.5-8.5; 8-12 mM magnesium chloride; 0.5-1.5 mM dithiothreitol.

7. The method of claim 4, wherein, The step of sticky end filling comprises: The enzyme-digested material is incubated at 60-65 ℃ for 15-25 minutes, and then cooled to 20-30 ℃; biotin-labeled bases and DNA polymerase are added to the enzyme-digested material, and incubated at 35-40 ℃ for 3-5 hours.

8. The method of claim 4, wherein, The step of intranuclear ligation comprises: A ligation reaction system containing DNA ligase is added to the end-filled material, and incubated at 15-18 ℃ for 3-5 hours and at 23-28 ℃ for 0.5-1.5 hours; The ligation reaction system containing DNA ligase comprises: 250-350 mM Tris-HCl, pH 7.5-8; 80-120 mM magnesium chloride; 80-120 mM dithiothreitol, and 0.5-1.5 mM ATP, 8-12 vol% Triton X-100, and 40-60 U T4 DNA ligase.

9. The method of claim 4, wherein, The step of DNA de-crosslinking comprises: The intranuclear ligation material is centrifuged at 800-1200 rcf for 2-4 minutes at 20-30 ℃, the supernatant is discarded, and resuspended with SDS lysis buffer; proteinase K is added, and incubated at 50-60 ℃ for 20-40 minutes; salt is added, and incubated at 60-70 ℃ for 8-12 hours; The SDS lysis buffer comprises: 40-60 mM Tris-HCl, pH 7.5-8.5, 0.5-1.5 vol% SDS, and 8-12 mM ethylenediaminetetraacetic acid.

10. The method of claim 1, wherein, The step of designing the RNA probe reverse complementary to the core promoter sequence comprises synthesizing the RNA probe reverse complementary to the core promoter sequence according to the plant core promoter library. The step of designing the RNA probe reverse complementary to the core promoter sequence comprises synthesizing the RNA probe reverse complementary to the core promoter sequence according to the plant core promoter library.

Images