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Method for improving accessibility of resolution chromatin of cells

A high-resolution, chromatin technology, applied in the field of improving cell resolution and chromatin accessibility, can solve the problems of high cost and low data resolution, and achieve the effect of low cost, cost reduction, and convenient sequencing on the machine

Inactive Publication Date: 2019-04-23
HUAZHONG AGRI UNIV
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Problems solved by technology

[0003] Compared with DNase-Seq, MNase-Seq, and FAIRE-Seq, the ATAC-Seq method is simpler and requires fewer cells. It only needs 500 to 50,000 fresh cells, and the operation time is shortened to several hours. However, due to the use of Tn5 Transposases lead to high costs, and the obtained data cannot be completely matched with DNase-Seq and MNase-Seq. It needs to be combined with ChIP-Seq and RNA-Seq for integrated analysis, and the resolution of the data is low (Buenrostro, Wu et al .2015)

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  • Method for improving accessibility of resolution chromatin of cells

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[0031] 1. Main reagents and their preparation methods

[0032] (1) Main reagents:

[0033]Water (Nuclease-Free Water) (Catalog #AM9932, Ambion, USA); Tris HCl pH7.5 (Catalog #AM9856, Ambion, USA); Sodium Chloride (Catalog #AM9760G, Ambion, USA); Magnesium Chloride (cat# AM9530G, Ambion, USA); Igepal CA-630 (Catalog #I8896, Sigma, USA); Phosphate Buffered Saline (PBS) (Catalog #344198, Millipore, USA); Qiagen MinElute PCR Product Purification Kit (Catalog #28004, QIAGEN, USA); polyethoxyethanol octylphenyl-polyethylene glycol (IGEPAL CA-630) (Cat. #13021, Sigma, USA); RPMI 1640 medium (Cat. #11875-093, Gibco, USA) ; Fetal bovine serum, Qualified, Australia Origin (Cat #10099-141, Gibco, USA); Non-essential aminoacid (Cat #11140050, life, USA)

[0034] (2) Preparation of reagents:

[0035] 1) The preparation of Lysis buffer lysis solution is shown in Table 1 (store at 4°C for one week after preparation):

[0036] Formulation: By mass: Tris·HCl pH 7.5 10mM; Sodium Chloride 10...

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Abstract

The invention belongs to the technical field of cell biology experiments, and particularly relates to a method for improving accessibility of resolution chromatin of cells. According to the method, based on a new NEBNext FS fragmented enzyme and a fragment selection strategy, system optimization and configuration are conducted pertinently, the cutting efficiency and precision of the chromatin aremaximized, finally, chromatin open region information with a high resolution is obtained, and meanwhile experimental costs are lowered greatly. A new technical route is provided for research on a chromatin open region, obtained data is high in quality, seamless connection with a standard program of second-generation library establishing sequencing can be further released, and the method is a convenience method for research on the accessibility of the chromatin.

Description

technical field [0001] The invention belongs to the technical field of cell biology experiments, and in particular relates to a method for improving cell resolution chromatin accessibility. Background technique [0002] At present, the research on 3D genome is becoming more and more popular, and the research topic of chromatin accessibility is one of the hot spots, which plays a crucial role in regulating gene expression (Buenrostro, Giresi et al. 2013). In eukaryotes, chromatin is tightly packaged into a series of nucleosomes, each nucleosome consists of histone octamers and DNA, chromatin in most genomes is tightly coiled within the nucleus, but There are also regions that are loosely remodeled by chromatin, a state or genetic structure known as chromatin accessibility. At present, there are many methods to study chromatin accessibility, such as: MNase-Seq, DNase-Seq, FAIRE-Seq, ATAC-Seq (Assay for Transposase-Accessible Chromatin with high-throughputsequencing), except M...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2531/113C12Q2521/313
Inventor 曹建华孙艳赵书红
Owner HUAZHONG AGRI UNIV
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