Whole genome interaction library and construction method thereof

A construction method and a genome-wide technology, applied in the field of high-throughput sequencing library construction, can solve cumbersome and time-consuming problems, and achieve the effect of wide operability

Active Publication Date: 2018-07-10
CHINA NAT RICE RES INST
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Problems solved by technology

[0009] The main purpose of the present invention is to provide a genome-wide interactive library and its constru

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  • Whole genome interaction library and construction method thereof
  • Whole genome interaction library and construction method thereof

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Embodiment 1

[0038] Choose 2g fresh leaves (approximately 25×10 6 Cells) were used as the starting material, and formaldehyde with a mass concentration of 1% was cross-linked at room temperature for 40-60 minutes to obtain cells with immobilized chromatin. The specific library construction steps were as follows:

[0039] 1) Cell lysis. Cells per copy (25×10 6 1) Add 1 ml of ice-cold lysate (10 mM Tris-HCL, 30 mM NaCl, 0.2% NP-40, 10% protease inhibitor, pH 7.4), and place on ice for 15 min. Submerge 4 groups of cells with a tissue homogenizer on ice, 30 times each. The lysate was centrifuged at 2,000xg for 5 min at room temperature. The samples were washed twice with pre-cooled restriction endonuclease corresponding digestion buffer (1×NEBuffer Cutsmart) and collected by centrifugation at 2000×g for 5 min. Resuspend the sample with 260 μL of this digestion buffer.

[0040] 2) Chromatin digestion. Add 1560 μL restriction endonuclease corresponding digestion buffer (1×NEBuffer Cutsmart...

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Abstract

The invention provides a whole genome interaction library and a construction method thereof. The construction method comprises the following steps of step S1, using a primer sequence to link ends, generated by enzyme digestion, of a DNA-protein cross-linking complex to form a cyclic complex; step S2, conducting decrosslinking on the cyclic complex to obtain cyclic DNA; step S3, conducting fragmented library construction on the cyclic DNA to obtain the whole genome interaction library. The method uses a section of exogenous known DNA sequence to link the ends of enzyme-digested product fragments, so that the DNA-protein cross-linking complex forms the cyclic complex, then proteins in the cyclic complex are removed by crosslinking to obtain the cyclic DNA, the exogenous known DNA sequence carried by the cyclic DNA is used for fragmented library construction to obtain the whole genome interaction library for whole genome interaction research, and a whole genome interaction research methodwhich is simple and has wide operability is provided.

Description

technical field [0001] The invention relates to the field of high-throughput sequencing library construction, in particular, to a genome-wide interaction library and a construction method thereof. Background technique [0002] Most of the early studies on the regulation of gene expression were based on the assumption that the DNA chain is a linear chain, that is, promoters, exons, introns, enhancers, insulators, etc. are distributed on the DNA chain in a linear form. In fact, eukaryotic DNA is stored in the nucleus in a highly folded and condensed form of chromatin. Chromatin has a high-level structure and conformation in space; and this high-level structure and conformation play a very important role in the process of gene transcription and regulation. Therefore, to thoroughly analyze the processes of gene transcription, DNA replication, and DNA repair, it is necessary to understand the spatial organization of chromatin, such as the spatial distribution of the genome, the ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C40B50/06C12Q1/6869C12Q1/6806
CPCC12N15/1093C12Q1/6806C12Q1/6869C40B50/06C12Q2535/122
Inventor 王克剑王春刘庆任俊
Owner CHINA NAT RICE RES INST
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