阅读说明：本技术 一种适用于海洋藻类的Hi-C高通量测序建库方法 (Hi-C high-throughput sequencing and database building method suitable for marine algae ) 是由 冯欢 于 2020-04-09 设计创作，主要内容包括：本发明提供的适用于海洋藻类的Hi-C高通量测序建库方法包括：1)海洋藻类样品甲醛交联；2)液氮研磨和裂解液并行裂解交联物料,释放并收集细胞染色质；3)percoll纯化细胞染色质；4)纯化后的染色质依次经SDS灭活内源酶、MboI核酸内切酶酶切打断获得酶切物料；5)酶切物料依次经粘性末端补平、分子内连接获得分子内连接物料；6)解交联分子内连接物料并纯化,打断纯化后的DNA成利于测序的片段大小从而获得DNA测序文库。该方法设计合适的细胞壁去除方法、细胞核纯化方法,使分析阶段背景噪音干扰显著减少,同时也解决了海洋藻类细胞壁厚难释放细胞质、海洋藻类样品建库效果差的技术问题,实现了海洋藻类的Hi-C高通量测序建库,扩大了Hi-C技术的适用范围。(The Hi-C high-throughput sequencing and database building method suitable for marine algae provided by the invention comprises the following steps of: 1) formaldehyde crosslinking of marine algae samples; 2) grinding with liquid nitrogen and cracking the crosslinked material with cracking liquid, and releasing and collecting cell chromatin; 3) percoll purified cellular chromatin; 4) the purified chromatin is subjected to SDS (sodium dodecyl sulfate) inactivation endogenous enzyme and MboI endonuclease digestion interruption in sequence to obtain a digestion material; 5) the enzyme digestion material is orderly subjected to viscous terminal filling and intramolecular connection to obtain an intramolecular connection material; 6) and (3) connecting materials in the uncrosslinked molecules, purifying, and breaking the purified DNA into fragments with sizes favorable for sequencing so as to obtain the DNA sequencing library. The method designs a proper cell wall removing method and a proper cell nucleus purifying method, so that the background noise interference in the analysis stage is obviously reduced, the technical problems that the wall thickness of marine algae cells is difficult to release cytoplasm and the marine algae sample library building effect is poor are solved, the Hi-C high-throughput sequencing library building of the marine algae is realized, and the application range of the Hi-C technology is expanded.)

1. A Hi-C high-throughput sequencing and banking method suitable for marine algae is characterized by comprising the following steps of:

1) performing formaldehyde crosslinking on a marine algae sample;

2) grinding with liquid nitrogen and cracking the crosslinked material with cracking liquid, and releasing and collecting cell chromatin;

3) percoll purified cellular chromatin;

4) the purified chromatin is subjected to SDS (sodium dodecyl sulfate) inactivation endogenous enzyme and MboI endonuclease digestion interruption in sequence to obtain a digestion material;

5) the enzyme digestion material is orderly subjected to viscous terminal filling and intramolecular connection to obtain an intramolecular connection material;

6) and (3) connecting materials in the uncrosslinked molecules, purifying, and breaking the purified DNA into fragments with sizes favorable for sequencing so as to obtain the DNA sequencing library.

2. The Hi-C high-throughput sequencing and banking method for marine algae according to claim 1, wherein the formaldehyde crosslinking step comprises: mixing the marine algae sample with the pre-cooled crosslinking system, placing on ice for vacuum incubation for 30-40min for formaldehyde crosslinking, and adding glycine to terminate crosslinking after incubation is finished.

3. The Hi-C high-throughput sequencing and banking method for marine algae according to claim 1, wherein the percoll purification step comprises:

1) 2M L2.5.5M sucrose, 5M L percoll solution and 1M L NIB re-suspended cell nucleus suspension are taken to be arranged in a centrifuge tube, wherein the percoll solution is diluted by NIB lysate, and the mass concentration is 30%;

2) centrifuging at 3000g for 30min, and collecting Percoll layer 3.5-6.5m L solution;

3) diluting the solution in the step 2) by 3-5 times with NIB lysate, centrifuging for 5min at 2500g, and discarding the supernatant.

4. The Hi-C high-throughput sequencing and banking method for marine algae according to claim 1, wherein the step of inactivating endogenous enzymes comprises:

1) resuspending the percoll purified pellet in 1.2 × NEBuffer2.1 at 100. mu. L, adding 1.5. mu. L20 wt% SDS per tube to dissolve chromatin, pipetting to mix well, resuspending all cell debris and avoiding foam formation;

2) the incubation was performed sequentially at 65 ℃ 900rpm for 10min, at 37 ℃ 900rpm for 30min, and immediately placed on ice and centrifuged instantaneously to remove the cap liquid.

5. The Hi-C high-throughput sequencing and banking method for marine algae according to claim 4, wherein the unreacted SDS of the inactivated endogenous enzyme is neutralized by Triton X-100 by adding 20 wt% Triton X-100 of 1 × NE buffer2.1 and 70 μ L of 400 μ L to the system of the inactivated endogenous enzyme obtained in step 2), resuspending cell debris, and shaking at 37 ℃ and 950rpm for 15 min.

6. The Hi-C high-throughput sequencing and banking method for marine algae according to claim 1, wherein the concentration of the MboI endonuclease is 5000units/m L, and the digestion reaction conditions are 900rpm and 37 ℃ for 4 hours.

7. A Hi-C high-throughput sequencing and library building method for marine algae according to claim 1, wherein the reaction system for the end filling in step 5) comprises the following components in an amount of digestion material 500. mu. L, NE buffer2.1(10 ×) 10. mu. L, 10mM dATP 2.0. mu. L, 10mM dGTP 2.0. mu. L, 10mM dTTP 2.0. mu. L, 5mM biotin-14-dCTP 4.0. mu. L, sterilized water 65. mu. L, and 5U/mu L Klenow polymerase 15. mu. L.

8. The Hi-C high-throughput sequencing and library building method suitable for marine algae according to claim 1, wherein the intramolecular ligation reaction system in the step 5) comprises 10% Triton X-1001m L, 10 × T4 ligation buffer 1m L, BSA (10mg/m L) 100 μ L, and H₂O 7.85mL、5U/μL T4 DNA ligase 50μL。

9. The Hi-C high-throughput sequencing and library building method suitable for marine algae according to claim 1, wherein the purification reaction system in step 6) comprises the following components in an amount of 1 μ g of uncrosslinked material, 10x NEBuffer2.110 μ L, 10mM dATP 1 μ L, 3U/μ L T4 DNA Polymerase 1.67 μ L, and 85.33 μ L.

10. The Hi-C high-throughput sequencing and library building method for marine algae according to claim 1, further comprising capturing the target fragment and library amplification.

Technical Field

The invention relates to the technical field of molecular biology, in particular to a Hi-C high-throughput sequencing and database building method suitable for marine algae.

Background

Chromosome Conformation Capture (3C) technology is a technology that studies Chromosome and protein interactions and Chromosome Conformation, can provide detailed information on the association between distant genetic loci, which can be captured from formaldehyde-fixed cell nuclei, and can be inferred from the three-dimensional folding pattern of chromosomes. In recent years, with the rapid development of second generation sequencing technologies, Hi-C derived from 3C technology is the subject of whole cell nucleus to study the association between gene loci in the whole genome. In the Hi-C technology, the relation of the whole chromatin DNA in the space position in the whole genome range is researched by taking the whole cell as a research object, utilizing a high-throughput sequencing technology and combining a bioinformatics method; the high-resolution chromatin three-dimensional structure information is obtained by capturing all DNA interaction modes in the chromatin. The Hi-C technology is widely applied and runs through the leading edge of current life science research and the hot field. The existing Hi-C technology makes minor modifications to the process of constructing 3C libraries. Specifically, the cohesive ends generated by the cleavage were filled with biotin-labeled nucleotides prior to ligation. After blunt end ligation, DNA was extracted and randomly broken using ultrasound, and finally biotin-labeled DNA fragments were captured to ensure that the data for subsequent analysis came more from a true interaction. After the DNA sequence pairs obtained by second-generation sequencing are aligned to a reference genome, if a pair of sequences corresponds to different n enzyme-digested fragments, the two fragments are considered to have n interactions, and thus a matrix of the connection frequency between all the enzyme-digested fragments in the whole genome can be constructed.

The conventional high-throughput plant Hi-C sequencing library is prepared by taking plant leaf tissues as research objects, the chromatin environment of the plant Hi-C sequencing library is relatively single compared with that of marine algae, better results can be obtained easily, but the application range of the plant Hi-C sequencing library is limited, and the plant Hi-C sequencing library is far away from the target of generally disclosing biological functions by research. Unlike leaf tissue, the complex cell wall structure of marine algae cannot release nuclei by simply lysing the cells. Meanwhile, experiments show that when the marine algae sample is processed by adopting the prior art, the cell nucleus cannot be completely released, and meanwhile, the subsequent enzyme digestion reaction effect is influenced by endogenous polysaccharide, polyphenol and mineral substances, so that the effectiveness of library data is influenced. Therefore, it is necessary to develop a Hi-C high-throughput sequencing and banking method suitable for marine algae.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides a Hi-C high-throughput sequencing and banking method suitable for marine algae through a large number of experimental contrastive analyses.

In order to realize the purpose, the invention is realized by the following technical scheme:

a Hi-C high-throughput sequencing and banking method suitable for marine algae comprises the following steps:

1) performing formaldehyde crosslinking on a marine algae sample;

2) grinding with liquid nitrogen and cracking the crosslinked material with cracking liquid, and releasing and collecting cell chromatin;

3) percoll purified cellular chromatin;

4) the purified chromatin is subjected to SDS (sodium dodecyl sulfate) inactivation endogenous enzyme and MboI endonuclease digestion interruption in sequence to obtain a digestion material;

5) the enzyme digestion material is orderly subjected to viscous terminal filling and intramolecular connection to obtain an intramolecular connection material;

6) and (3) connecting materials in the uncrosslinked molecules, purifying, and breaking the purified DNA into fragments with sizes favorable for sequencing so as to obtain the DNA sequencing library.

Further, the formaldehyde crosslinking step comprises: mixing the marine algae sample with the pre-cooled crosslinking system, placing on ice for vacuum incubation for 30-40min for formaldehyde crosslinking, and adding glycine to terminate crosslinking after incubation is finished.

Further, the percoll purification step is as follows:

1) 2M L2.5.5M sucrose, 5M L percoll solution and 1M L NIB re-suspended cell nucleus suspension are taken to be arranged in a centrifuge tube, wherein the percoll solution is diluted by NIB lysate, and the mass concentration is 30%;

2) centrifuging at 3000g for 30min, and collecting Percoll layer 3.5-6.5m L solution;

3) diluting the solution in the step 2) by 3-5 times with NIB lysate, centrifuging for 5min at 2500g, and discarding the supernatant.

Further, the step of inactivating the endogenous enzyme comprises:

1) resuspending the percoll purified pellet in 1.2 × NEBuffer2.1 at 100. mu. L, adding 1.5. mu. L20 wt% SDS per tube to dissolve chromatin, pipetting to mix well, resuspending all cell debris and avoiding foam formation;

2) the incubation was performed sequentially at 65 ℃ 900rpm for 10min, at 37 ℃ 900rpm for 30min, and immediately placed on ice and centrifuged instantaneously to remove the cap liquid.

Further, the unreacted SDS to inactivate the endogenous enzyme was neutralized with Triton X-100 by adding 400. mu. L of 1 × NEBuffer2.1 and 70. mu. L of 20 wt% Triton X-100 to the system of inactivated endogenous enzyme obtained in step 2), resuspending the cell debris, and shaking at 37 ℃ and 950rpm for 15 min.

Further, the concentration of the MboI endonuclease was 5000units/m L, and the digestion reaction was carried out at 900rpm and 37 ℃ for 4 hours.

Further, the reaction system for the end-filling in step 5) comprises the following components in amounts of digestion material 500. mu. L, NE Buffer2.1(10 ×) 10. mu. L, 10mM dATP 2.0. mu. L, 10mM dGTP 2.0. mu. L, 10mM dTTP 2.0. mu. L, 5mM biotin-14-dCTP 4.0. mu. L, sterilized water 65. mu. L, and 5U/. mu. L Klenow polymerase 15. mu. L.

Further, the intramolecular ligation reaction system in the step 5) comprises the following components in the dosage of 10% Triton X-1001m L, 10 × T4 ligationbuffer 1m L, BSA (10mg/m L) 100 mu L, H₂O 7.85mL、5U/μLT4DNAligase 50μL。

Further, the purification reaction system in the step 6) comprises the following components in the following amounts of 1. mu.g of uncrosslinked material, 10 XNEBuffer2.110. mu. L, 10mM dATP 1. mu. L, 3U/. mu. L T4 DNApolymerase 1.67. mu. L and water 85.33. mu. L, and the purification reaction conditions are that the reaction is carried out at 12 ℃ for 4h and the reaction is stopped by 2. mu. L0.5.5M EDTA.

Further, the method further comprises capturing the fragment of interest and amplifying the library.

Compared with the prior art, the invention has the beneficial effects that:

(1) the Hi-C high-throughput sequencing and library building method for marine algae provided by the invention is designed aiming at the special structure of marine algae cells, so that the background noise interference in the analysis stage is obviously reduced, the technical problems that the wall thickness of the marine algae cells is difficult to release cytoplasm and the library building effect of marine algae samples is poor are solved, the Hi-C high-throughput sequencing and library building of the marine algae are realized, and the application range of the Hi-C technology is expanded. The library construction method is simple and convenient in operation process and can be copied to other laboratories with molecular biology bases.

(2) Compared with the traditional Hi-C method, the method for constructing the library has the advantages that the step of purifying the cell nucleus by percoll is added, the purity of the cell nucleus is obviously improved, the influence of polysaccharide, polyphenol and mineral substances on the enzyme activity in the Hi-C library construction process is reduced, and the method can obviously improve the influence of impurities on the experimental result and the analysis process.

(3) In the step of cell wall cracking, the database construction method obtains the wall-breaking conditions with good cell wall cracking effect and relatively good genome integrity through gradient condition exploration.

(4) Compared with the traditional Hi-C method, the database building method has the advantages that the reaction time is obviously shortened and the inactivation effect is improved in the steps of endogenous enzyme inactivation and SDS neutralization; particularly aiming at the marine algae sample with high impurity content, the method can obviously reduce the influence of more impurities on the experimental result and the analysis process; the inactivation system of the endogenous enzyme adopts the principle of small system inactivation and large system neutralization, so that the inactivation effect of the sample is improved;

(5) in the aspect of selection of tool enzyme, the MboI with wide applicability is selected, the NEBBuffer 2.1 can be used in the whole reaction system, the step of frequently replacing a buffer solution system in the traditional Hi-C method is omitted, the operation steps are greatly simplified, the MboI is the four-base enzyme, and the enzyme cutting resolution is improved compared with the six-base enzyme in the traditional method.

(6) The Hi-C library sequencing data constructed by the invention can be used for not only the research of gene expression regulation, but also the auxiliary genome assembly.

Drawings

FIG. 1 is a flow chart of Hi-C high throughput sequencing and database building method for marine algae according to the present invention.

FIG. 2 is the agarose gel electrophoresis pattern of the example 1 for identifying the integrity and the digestion effect of the laver genome.

FIG. 3 is a diagram showing the size distribution results of the laver library constructed in example 1.

FIG. 4 is an agarose gel electrophoresis pattern of comparative example for identifying the integrity of laver genome and the effect of enzyme digestion by using the conventional Hi-C method.

Detailed Description

The following examples are presented to illustrate certain embodiments of the invention in particular and should not be construed as limiting the scope of the invention. The present disclosure may be modified from materials, methods, and reaction conditions at the same time, and all such modifications are intended to be within the spirit and scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.