ATAC-seq
(Assay for Transposase-Accessible Chromatin with high throughput sequencing)
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技术概述

ATAC-seq(Assay for Transposase-Accessible Chromatin with high throughput sequencing) 是2013年由斯坦福大学William J. Greenleaf和Howard Y. Chang实验室开发的用于研究染色质可及性(通常也理解为染色质的开放性)的方法, 原理是通过转座酶Tn5容易结合在开放染色质的特性,然后对Tn5酶捕获到的DNA序列进行测序。
真核生物的核DNA并不是裸露的,而是与组蛋白结合形成染色体的基本结构单位核小体,核小体再经逐步的压缩折叠最终形成染色体高级结构(如人的DNA链完整展开约2m长,经过这样的折叠就变成了纳米级至微米级的染色质结构而可以储存在小小的细胞核)。而DNA的复制转录是需要将DNA的紧密结构打开,从而允许一些调控因子结合(转录因子或其他调控因子)。这部分打开的染色质,就叫开放染色质,打开的染色质允许其他调控因子结合的特性称为染色质的可及性(chromatin accessibility)。因此,认为染色质的可及性与转录调控密切相关。
开放染色质的研究方法有ATAC-seq以及传统的DNase-Seq及FAIRE-seq等,ATAC-Seq由于所需细胞量少,实验简单,可以在全基因组范围内检测染色质的开放状态,目前已经成为研究染色质开放性的首选技术方法。

图片来源:Nat Methods, 2013. doi: 10.1038/nmeth.2688. Epub 2013 Oct.
(资料来源:简书-六六_ryx)

技术详情

ATAC-Seq、ChIP-Seq、Dnase-Seq、MNase-Seq、FAIRE-Seq整体的分析思路一致,找到富集区域,对富集区域进行功能分析。
• ChIP-Seq是揭示特定转录因子或蛋白复合物的结合区域,实际是研究DNA和蛋白质的相互作用,利用抗体将蛋白质和DNA一起富集,并对富集到的DNA进行测序。
• DNase-Seq、ATAC-Seq、FAIRE-Seq都是用来研究开放染色质区域。DNase-Seq是用的DNase I内切酶识别开放染色质区域,而ATAC-seq是用的Tn5转座酶,随后进行富集和扩增;FAIRE-Seq是先进行超声裂解,然后用酚-氯仿富集。
• MNase-Seq是用来鉴定核小体区域。

图片来源:https://cmb.i-learn.unito.it/mod/wiki/view.php?pageid=78

Figure 1. Overview of ChIP-seq, DNase-seq, ATAC-seq and MNase-seq experiments.

A genomic locus analyzed by complementary chromatin profiling experiments reveals different facets of chromatin structure; ChIP-seq reveals binding sites of specific transcription factors, DNase-seq and ATAC-seq reveal regions of open chromatin while MNase-seq identifies well-positioned nucleosomes. In ChIP-seq chromatin immunoprecipitation (ChIP) is used to extract DNA fragments that are bound to the target protein, either directly or via other proteins in a complex containing the target factor. In DNase-seq, chromatin is lightly digested by the DNase I endonuclease. Size selection is used to enrich for fragments that are produced in regions of chromatin where the DNA is highly sensitive to DNase I attack. ATAC-seq is an alternative to DNase-seq that uses an engineered Tn5 transposase to cleave DNA and to integrate primer DNA sequences into the cleaved genomic DNA. Micrococcal nuclease (MNase) is an endo-exo- nuclease that processively digests DNA until an obstruction such as a nucleosome is reached.[ ]

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参考文献

  • [[1]] Mayer, C.A., Liu, X.S. Identifying and Mitigating Bias in Next-Generation Sequencing Methods for Chromatin Biology. Nat Rev Genet. 2014. 15(11): 709–721.

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