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Library construction method applicable to cell line and animal tissue ATAC-seq sequencing technology

A technology for animal tissue and library construction, applied in the field of molecular biology, can solve the problems of high price, high manpower, and unsuitability for large-throughput operations of fluorescence microscopes, and achieve the effect of wide applicability and simple operation

Inactive Publication Date: 2019-06-18
嘉兴菲沙基因信息有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First of all, the price of fluorescence microscope is very high, many laboratories do not have this instrument, and DAPI is a carcinogenic reagent, and the use of microscope counting requires a lot of manpower, which is not suitable for large-throughput operations

Method used

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  • Library construction method applicable to cell line and animal tissue ATAC-seq sequencing technology
  • Library construction method applicable to cell line and animal tissue ATAC-seq sequencing technology
  • Library construction method applicable to cell line and animal tissue ATAC-seq sequencing technology

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Embodiment 1

[0031] Aiming at the particularity of cell lines and animal tissues, the present invention proposes a library construction method suitable for ATAC-seq sequencing technology of cell lines and animal tissues, said method comprising the following steps:

[0032] 1. Collect cells or animal tissues and prepare cell suspension

[0033] 1.1 Take a sufficient amount of cell lines or animal tissues, and the preservation and processing methods for frozen cell lines and tissues are as follows:

[0034] (1) The composition of the cell line is relatively simple, and the animal cell line does not contain a cell wall, and the cell membrane is easily damaged during quick freezing. Therefore, the method of slow freezing and rapid thawing of the cryopreservation solution is used, and the recovered cell line is cooled at 4°C for 30 minutes and then cooled at -20°C. After freezing for 2 hours, store at -80°C for long-term storage. When starting the experiment, thaw quickly at 37°C, centrifuge at...

Embodiment 2

[0063] 1. Library quality inspection

[0064] Such as figure 1 As shown, the DNA that wraps around the nucleosome can be seen as two parts, the part that wraps around the nucleosome has a length of 146 bp, and there is a partially exposed fragment σ with an indeterminate length.

[0065] According to the ATAC-seq experiment of Example 1, the fragment (about 60bp) between the nucleosomes plus the joint of 136bp is obtained as the first peak of a total of about 200bp; fragments. DNA winding nucleosome fragment size is about 146bp, with some naked fragments in the middle, and finally a 136bp linker, forming a second peak at about 300-400bp; two nucleosome winding DNA plus linker formation The third peak presents a phenomenon unique to ATAC-seq experiments. The result is as figure 2 As shown, peak 1 is a fragment between nucleosomes, about 60bp, plus a 136bp linker is about 200bp, and peak 2 is a fragment 146bp plus a 136bp linker wrapped around a nucleosome plus a part of th...

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Abstract

The invention relates to a library construction method applicable to a cell line and animal tissue ATAC-seq sequencing technology. The method comprises steps as follows: 1) cell or animal tissue is collected, and a cell suspension is prepared; 2) a lysate containing NP40 is added to split cell membranes, and cell nucleuses are obtained and purified; 3) the cell nucleuses are collected for quantitative and integrity detection; 4) Tn5 transposase is added to complete cell nucleuses for a transposition reaction and purification; 5) PCR amplification is performed, and a second library is constructed and purified; 6) high-throughput sequencing is performed, and gene data are obtained. According to the method, quantitative operation is performed directly through the DNA content on the basis of the characteristic that transposase is sensitive to DNA content, and the method is wide in applicability and particularly applicable to library construction of rare species or tissue and tissue in poorpreservation state. Meanwhile, the recycled cell nucleuses can be used for Tn5 transposase developed by domestic vazyme company through early optimization, good experiment results are obtained, and ATAC-seq is not limited by the variety of transposase any more.

Description

technical field [0001] The present invention relates to the technical field of molecular biology, and more specifically, relates to a library construction method suitable for ATAC-seq sequencing technology of cell lines and animal tissues. Background technique [0002] The nucleosome is the basic structural unit of eukaryotic chromatin, composed of DNA and histones. Each nucleosome is formed by winding 146bp DNA on octamer histone, and the two nucleosomes are connected by a piece of connecting DNA, and the combination of DNA and histone can change dynamically. DNA wrapped around histones is not easy to bind to other proteins, and these DNAs are usually in a state of expression repression. Open DNA regions without nucleosome binding are easy to bind to regulatory proteins, which can make their downstream genes active. Cells regulate the expression of genes by changing the binding positions of DNA and histones. Therefore, obtaining the DNA sequence in an open state can be u...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6869C40B50/06
Inventor 严琰
Owner 嘉兴菲沙基因信息有限公司
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