阅读说明：本技术 冰冻植物组织Hi-C建库的方法 (Method for building frozen plant tissue Hi-C library ) 是由 李彪 郭松 仉旭 沈世超 于 2020-08-14 设计创作，主要内容包括：本发明公开了一种冰冻植物组织Hi-C建库的方法。该方法包括以下步骤：S1,冰冻植物组织液氮研磨；S2,加入甲醛进行固定,加入甘氨酸进行终止固定；S3,过细胞筛网；S4,将固定化的细胞进行细胞裂解,然后进行限制性内切酶消化；S5,进行标记物标记,并平末端化；S6,重新连接；S7,解除固定化,进行DNA片段的提取,回收DNA片段；S8,去除带有标记物标记但未连接的DNA片段；S9,DNA打断；S10,钓取含有标记物标记的DNA片段；S11,末端修复加接头；S12,PCR扩增,获得Hi-C文库。应用本发明的建库的方法,利用冰冻植物组织进行Hi-C建库,可以达到与新鲜叶片Hi-C建库同等的有效数据率。(The invention discloses a method for establishing a bank of frozen plant tissues Hi-C. The method comprises the following steps: s1, grinding the frozen plant tissues by liquid nitrogen; s2, adding formaldehyde for fixation, and adding glycine for termination of fixation; s3, passing through a cell screen; s4, performing cell lysis on the immobilized cells, and then performing restriction enzyme digestion; s5, marking a marker, and flattening and terminating; s6, reconnection; s7, removing immobilization, extracting DNA fragments, and recovering the DNA fragments; s8, removing the DNA fragment which is marked by the marker and is not connected; s9, breaking DNA; s10, fishing out a DNA fragment containing the marker; s11, repairing the tail end and adding a joint; s12, PCR amplification, obtaining Hi-C library. By applying the method for establishing the banks, Hi-C bank establishment is carried out by using frozen plant tissues, and the effective data rate which is the same as that of Hi-C bank establishment of fresh leaves can be achieved.)

1. A method for establishing a frozen plant tissue Hi-C library is characterized by comprising the following steps:

s1, grinding the frozen plant tissues by liquid nitrogen;

s2, adding formaldehyde into the ground plant tissues for fixation, and then adding glycine for termination of fixation to obtain a fixed product;

s3, passing the fixed product through a cell screen, and centrifuging to obtain fixed cells;

s4, performing cell lysis on the immobilized cells, and then performing restriction enzyme digestion to obtain immobilized chromosome fragments;

s5, labeling the ends of the immobilized chromatin fragments with a label, and performing blunt-end to obtain blunt-end immobilized chromatin fragments;

s6, religation of the immobilized chromatin fragments with flat ends to obtain religated immobilized chromatin fragments;

s7, the religation of the immobilized chromatin fragments is released, the extraction of DNA fragments is carried out, and the DNA fragments are recovered;

s8, removing the DNA fragment which is marked by the marker and is not connected with the DNA fragment recovered in the S7 to obtain a purified DNA fragment;

s9, breaking DNA of the purified DNA fragment obtained in the S8;

s10, obtaining a DNA fragment containing a label of the labeling substance by using a magnetic bead having an affinity for the labeling substance;

s11, carrying out end repair and joint on the DNA fragment marked by the marker obtained in S10;

s12, carrying out PCR amplification on the product obtained in the S11 to obtain a Hi-C library.

2. The method according to claim 1, wherein the volume concentration of the formaldehyde in the S2 is 5-10%, the fixing temperature is room temperature, and the fixing time is 30-60 min.

3. The method according to claim 1, wherein the concentration of glycine in S2 is 1.5-3M; preferably 2.5M.

4. The method according to claim 1, wherein the plant tissue ground in S1 is added with a phosphate buffer.

5. The method of any one of claims 1 to 4, wherein the marker is biotin; the magnetic beads capable of being compatible with the label are streptavidin magnetic beads.

6. The method according to any one of claims 1 to 4, wherein in S4, the cells are lysed with 0.1% -1% SDS at 37 ℃ for 10 min; then adding Triton X-100 with a final concentration of 1% -3% at 37 deg.C for 30min, and neutralizing SDS.

7. The method of any one of claims 1 to 4, wherein in S4 the restriction enzyme is DnpII.

8. The method according to any one of claims 1 to 4, wherein the frozen plant tissue is frozen plant leaves.

9. The method according to claim 1, wherein the fragment size after fragmentation in S9 is 350-500 bp.

10. The method according to claim 1, characterized in that it comprises in particular the steps of:

weighing 1-3 g of frozen plant leaves, putting the frozen plant leaves into a precooled mortar, and carrying out liquid nitrogen grinding;

transferring the leaves ground by liquid nitrogen into a 50ml centrifuge tube, adding 30ml of phosphate buffer solution, then adding formaldehyde with the final concentration of 5-10%, and fixing for 60min at room temperature;

adding 2.04mL of 2.5M glycine, and stopping fixing at room temperature for 5 min;

passing the fixed product through a cell screen, collecting filtrate, and centrifuging to obtain the immobilized cells;

adding SDS with the final concentration of 0.1-1% into the immobilized cells for cell lysis, and carrying out 10min at 37 ℃; then adding Triton X-100 with the final concentration of 1-3%, neutralizing SDS at 37 ℃ for 30min to obtain a neutralized product;

adding 200U DnpII into the neutralization product for enzyme digestion, and obtaining an immobilized chromosome fragment under the enzyme digestion condition of 37 ℃ overnight;

carrying out biotin labeling on the tail end of the immobilized chromatin fragment, and flattening the tail end to obtain a flattened tail end immobilized chromatin fragment; the biotin label is dATP, the used terminal repair enzyme is Klenow Fragment, the reaction temperature is 37 ℃, and the reaction time is 45 min;

religation of the blunt-ended immobilized chromatin fragments to obtain religated immobilized chromatin fragments; the ligase used is T4 DNA ligase, the reaction temperature is 20 ℃, and the reaction time is 30 min;

adding protease K and SDS, digesting protein, removing immobilization, reacting at 65 deg.C for 90 min; after protein digestion, adding magnetic beads to extract DNA fragments, and recovering the DNA fragments;

removing the DNA fragment which carries the biotin label and is not connected from the recovered DNA fragment to obtain the purified DNA fragment, wherein the enzyme is T4 DNA polymerase, and the reaction condition is 12 ℃ for 2 h;

breaking the purified DNA by using an ultrasonic breaking physical method, wherein the size of the broken fragment is 350-500 bp;

utilizing streptavidin magnetic beads to hook DNA fragments containing biotin labels, wherein the reaction condition is 20 ℃ for 20 min;

performing end repair and adding a joint on the obtained biotin-labeled DNA fragment;

and performing PCR amplification to obtain the Hi-C library.

Technical Field

The invention relates to the technical field of biology, in particular to a method for establishing a bank of frozen plant tissues Hi-C.

Background

DNA is a carrier of genetic information of cells, exists in the form of chromatin in each cell in an organism, and controls the progress of the whole life activity. Most of the studies on DNA information have been conducted to study the sequence of bases in a DNA molecule (one-dimensional information of DNA), and the rules of life activities have been studied by analyzing the information on the base arrangement.

The nucleus in the real state is a narrow three-dimensional space, the DNA with a linear molecular structure can be positioned in the nucleus in a complex curling mode, the original one-dimensional DNA sequence is endowed with a three-dimensional space conformation, and a large number of complex gene regulation action modes are caused. In this regard, simple one-dimensional DNA sequence information cannot provide information about the spatial distribution of true DNA, and therefore cannot explain a series of gene regulation phenomena due to spatial conformation. To solve this problem, a series of detection methods are available. Such as the 3c (chromosome structure capture) method and the derivative 4c, 5c method. The methods all use sequencing as a basic detection means, utilize proteins in cell nuclei to form DNA structure fixed factors, construct DNA sequences with spatial structure information by fragment reconnection of DNA and the like, finally use a sequencing technology to detect chromatin DNA information, and calculate the distribution and interaction of the chromatin DNA information in space. Although such methods are capable of providing partial chromatin interaction information to some extent. However, due to the limitations of the methods and technologies, the methods only can detect a fixed point or a part of DNA interaction sites, and cannot explore the stereo interaction information at the level of the whole nucleus. And therefore inevitably a large amount of information will be missed. This is particularly important in the discovery of unknown interaction information.

With the advent of high throughput sequencing technologies in recent years, the availability of large-scale genomic information has become easier. The Hi-C technology is a technology for detecting information of chromatin in the whole cell nucleus by combining a high-throughput sequencing method. The Hi-C technology is a derivative technology of Chromosome conformation capture (Chromosome conformation capture, referred to as 3C for short), and refers to capture of Chromosome conformation based on high throughput, which can capture spatial interaction between different gene loci in the whole genome range, and study DNA elements of regulatory genes in three-dimensional space.

For example, Genome-wide analysis of local chromatography packing in Arabidopsis thaliana (2015) reported a Hi-C banking method of fresh Arabidopsis seedlings, which utilizes formaldehyde to fix chromatin structure, then breaks the original genomic sequence by restriction endonuclease, and carries out biotin labeling, and then rejoins to form a new DNA molecule with structural information. In this process, if two DNA molecule fragments at different genomic positions are joined to form a hybrid molecule, this is considered evidence that the two DNA molecules are in spatial proximity to each other. Then purifying and crushing the DNA, fishing the marked biotin molecules, and enriching to obtain the required DNA heterozygote molecules with spatial interaction. And finally constructing a library of high-throughput sequencing and double-end sequencing detection to obtain the spatial interaction information of the holochromatin.

However, the frozen plant leaf Hi-C constructed by the method reported in the literature fails to be finally constructed. In addition, the existing fresh plant leaf fixing step is not suitable for frozen plant leaves, the fixing effect is poor, samples are easy to degrade, and finally the failure of building a warehouse is caused.

Disclosure of Invention

The invention aims to provide a method for establishing a bank by freezing plant tissues Hi-C, which aims to solve the technical problem that the bank establishment of the frozen plant leaves Hi-C fails in the prior art.

In order to achieve the above object, according to one aspect of the present invention, there is provided a method of banking frozen plant tissues Hi-C. The method comprises the following steps: s1, grinding the frozen plant tissues by liquid nitrogen; s2, adding formaldehyde into the ground plant tissues for fixation, and then adding glycine for termination of fixation to obtain a fixed product; s3, passing the fixed product through a cell screen, and centrifuging to obtain the immobilized cell; s4, performing cell lysis on the immobilized cells, and then performing restriction enzyme digestion to obtain immobilized chromosome fragments; s5, labeling the ends of the immobilized chromatin fragments with a labeling substance, and performing blunt-end to obtain blunt-end immobilized chromatin fragments; s6, religation of the immobilized chromatin fragments with flat terminal ends to obtain religated immobilized chromatin fragments; s7, the immobilization of the newly connected immobilized chromatin fragments is released, the extraction of DNA fragments is carried out, and the DNA fragments are recovered; s8, removing the DNA fragment which is marked by the marker and is not connected with the DNA fragment recovered in the S7 to obtain a purified DNA fragment; s9, breaking DNA of the purified DNA fragment obtained in S8; s10, using magnetic beads capable of being compatible with the marker to catch the DNA fragment containing the marker; s11, and carrying out end repair and joint addition on the DNA fragment marked by the marker obtained in S10; s12, carrying out PCR amplification on the product obtained in the S11 to obtain a Hi-C library.

Further, the volume concentration of the formaldehyde in the S2 is 5% -10%, the fixing temperature is room temperature, and the fixing time is 30-60 min.

Further, the concentration of glycine in S2 is 1.5-3M; preferably 2.5M. 4.1 method, adding phosphate buffer to the ground plant tissue in S1.

Further, the marker is biotin; the magnetic beads capable of being compatible with the label are streptavidin-based magnetic beads.

Further, in S4, 0.1% -1% SDS is adopted for cell lysis, the temperature is 37 ℃, and the time is 10 min; then adding Triton X-100 with a final concentration of 1% -3% at 37 deg.C for 30min, and neutralizing SDS.

Further, in S4, the restriction enzyme is DnpII.

Further, the frozen plant tissue is frozen plant leaves.

Furthermore, in S9, the size of the fragmented fragment was 350-500 bp.

Further, the method specifically comprises the following steps: weighing 1-3 g of frozen plant leaves, putting the frozen plant leaves into a precooled mortar, and carrying out liquid nitrogen grinding; transferring the leaves ground by liquid nitrogen into a 50ml centrifuge tube, adding 30ml of phosphate buffer solution, then adding formaldehyde with the final concentration of 5-10%, and fixing for 60min at room temperature; adding 2.04mL of 2.5M glycine, and stopping fixing at room temperature for 5 min; passing the fixed product through a cell screen, collecting filtrate, and centrifuging to obtain immobilized cells; adding SDS with final concentration of 0.1-1% into the immobilized cells for cell lysis, and carrying out 10min at 37 ℃; then adding Triton X-100 with the final concentration of 1-3%, neutralizing SDS at 37 ℃ for 30min to obtain a neutralized product; adding 200U DnpII into the neutralized product for enzyme digestion, and obtaining an immobilized chromosome fragment under the enzyme digestion condition of 37 ℃ overnight; carrying out biotin labeling on the tail end of the immobilized chromatin fragment, and flattening the tail end to obtain a flattened tail end immobilized chromatin fragment; the biotin label is dATP, the used terminal repair enzyme is Klenow Fragment, the reaction temperature is 37 ℃, and the reaction time is 45 min; religation of the immobilized chromatin fragments with blunt ends to obtain religated immobilized chromatin fragments; the ligase used is T4 DNA ligase, the reaction temperature is 20 ℃, and the reaction time is 30 min; adding protease K and SDS, digesting protein, removing immobilization, reacting at 65 deg.C for 90 min; after protein digestion, adding magnetic beads to extract DNA fragments, and recovering the DNA fragments;

removing the DNA fragment which is not connected and is provided with the biotin label from the recovered DNA fragment to obtain a purified DNA fragment, wherein the used enzyme is T4 DNA polymerase, and the reaction condition is 12 ℃ for 2 h;

breaking the purified DNA by using an ultrasonic breaking physical method, wherein the size of the broken fragment is 350-500 bp;

utilizing streptavidin magnetic beads to hook DNA fragments containing biotin labels, wherein the reaction condition is 20 ℃ for 20 min;

carrying out end repair and adding a joint on the obtained biotin-labeled DNA fragment;

and performing PCR amplification to obtain the Hi-C library.

By applying the method for establishing the banks, Hi-C bank establishment is carried out by using frozen plant tissues, so that the effective data rate which is the same as that of Hi-C bank establishment of fresh leaves can be achieved; experiments prove that frozen corn, wheat, arabidopsis, cucumber, cotton and rape leaves are successfully built, the effective data rate is more than 25%, and the level of building a fresh leaf Hi-C library is achieved.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.

At present, the method for establishing the Hi-C plant sample library must use fresh plant leaves to establish the library, and if the frozen plant leaves are used to establish the library, the leaves are not easy to fix and are easy to degrade in the fixing process, and finally the library establishment fails. However, in the practical application process, fresh samples cannot be adopted for part of forest/medicinal material/wild plant samples, and a lot of frozen plant samples with long storage time (1 month to half year) are required to be built. In order to solve the technical problem, the invention provides the following technical scheme.

According to an exemplary embodiment of the present invention, a method for banking frozen plant tissues Hi-C is provided. The method comprises the following steps: s1, grinding the frozen plant tissues by liquid nitrogen; s2, adding formaldehyde into the ground plant tissues for fixation, and then adding glycine for termination of fixation to obtain a fixed product; s3, passing the fixed product through a cell screen, and centrifuging to obtain the immobilized cell; s4, performing cell lysis on the immobilized cells, and then performing restriction enzyme digestion to obtain immobilized chromosome fragments; s5, labeling the ends of the immobilized chromatin fragments with a labeling substance, and performing blunt-end to obtain blunt-end immobilized chromatin fragments; s6, religation of the immobilized chromatin fragments with flat terminal ends to obtain religated immobilized chromatin fragments; s7, the immobilization of the newly connected immobilized chromatin fragments is released, the extraction of DNA fragments is carried out, and the DNA fragments are recovered; s8, removing the DNA fragment which is marked by the marker and is not connected with the DNA fragment recovered in the S7 to obtain a purified DNA fragment; s9, breaking DNA of the purified DNA fragment obtained in S8; s10, using magnetic beads capable of being compatible with the marker to catch the DNA fragment containing the marker; s11, and carrying out end repair and joint addition on the DNA fragment marked by the marker obtained in S10; s12, carrying out PCR amplification on the product obtained in the S11 to obtain a Hi-C library.

By applying the method for establishing the banks, disclosed by the invention, Hi-C banks are established by utilizing frozen plant tissues, so that the effective data rate which is equal to that of Hi-C banks established by fresh leaves can be achieved

Preferably, the volume concentration of formaldehyde in S2 is 5% -10%, the fixing temperature is room temperature, the fixing time is 30-60 min, the integrity of cell nuclei can be kept by the fixing, the sample degradation is avoided, the random connection of later-stage library building is reduced, and the noise is reduced. Preferably, the concentration of glycine in S2 is 1.5-3M, more preferably 2.5M.

In a typical embodiment of the present invention, phosphate buffer is added to the ground plant tissue in S1.

The label in the present invention is for the purpose of subsequent DNA isolation, and for example, the label may be biotin, and the magnetic beads having affinity for the label may be streptavidin-modified magnetic beads. Preferably, in the cell lysis, 0.1% -1% SDS is adopted for cell lysis, the temperature is 37 ℃, and the time is 10 min; then adding Triton X-100 with a final concentration of 1% -3% at 37 deg.C for 30min, and neutralizing SDS. In the present invention, the restriction enzyme may be HindIII, MboI, AatII, ClaI, DnpII, etc., preferably DnpII.

Typically, the frozen plant tissue is frozen plant leaves. In S9, the size of the fragmented fragment is 350-500bp, so that subsequent sequencing and analysis are facilitated, and the sequencing quality is improved; because the fragments are too long, the quality of sequencing is reduced, and too short a fragment affects mapping rate.

In a preferred embodiment of the present invention, the method specifically comprises the steps of:

weighing 1-3 g of frozen plant leaves, putting the frozen plant leaves into a precooled mortar, and carrying out liquid nitrogen grinding;

transferring the leaves ground by liquid nitrogen into a 50ml centrifuge tube, adding 30ml of phosphate buffer solution, then adding formaldehyde with the final concentration of 5-10%, and fixing for 60min at room temperature;

adding 2.04mL of 2.5M glycine, and stopping fixing at room temperature for 5 min;

passing the fixed product through a cell screen, collecting filtrate, and centrifuging to obtain immobilized cells;

adding SDS with final concentration of 0.1-1% into the immobilized cells for cell lysis, and carrying out 10min at 37 ℃; then adding Triton X-100 with the final concentration of 1-3%, neutralizing SDS at 37 ℃ for 30min to obtain a neutralized product;

adding 200U DnpII into the neutralized product for enzyme digestion, and obtaining an immobilized chromosome fragment under the enzyme digestion condition of 37 ℃ overnight;

carrying out biotin labeling on the tail end of the immobilized chromatin fragment, and flattening the tail end to obtain a flattened tail end immobilized chromatin fragment; the biotin label is dATP, the used terminal repair enzyme is Klenow Fragment, the reaction temperature is 37 ℃, and the reaction time is 45 min;

religation of the immobilized chromatin fragments with blunt ends to obtain religated immobilized chromatin fragments; the ligase used is T4 DNA ligase, the reaction temperature is 20 ℃, and the reaction time is 30 min;

adding protease K and SDS, digesting protein, removing immobilization, reacting at 65 deg.C for 90 min; after protein digestion, adding magnetic beads to extract DNA fragments, and recovering the DNA fragments;

removing the DNA fragment which is not connected and is provided with the biotin label from the recovered DNA fragment to obtain a purified DNA fragment, wherein the used enzyme is T4 DNA polymerase, and the reaction condition is 12 ℃ for 2 h;

breaking the purified DNA by using an ultrasonic breaking physical method, wherein the size of the broken fragment is 350-500 bp;

utilizing streptavidin magnetic beads to hook DNA fragments containing biotin labels, wherein the reaction condition is 20 ℃ for 20 min;

carrying out end repair and adding a joint on the obtained biotin-labeled DNA fragment;

and performing PCR amplification to obtain the Hi-C library.

The following examples are provided to further illustrate the advantageous effects of the present invention.