ATAC-seq
(Assay for Transposase-Accessible Chromatin with high throughput sequencing)
技術概述
ATAC-seq(Assay for Transposase-Accessible Chromatin with high throughput sequencing) 是2013年由斯坦福大學William J. Greenleaf和Howard Y. Chang實驗室開發(fā)的用于研究染色質可及性(通常也理解為染色質的開放性)的方法, 原理是通過轉座酶Tn5容易結合在開放染色質的特性,然后對Tn5酶捕獲到的DNA序列進行測序。
真核生物的核DNA并不是裸露的,而是與組蛋白結合形成染色體的基本結構單位核小體,核小體再經(jīng)逐步的壓縮折疊最終形成染色體高級結構(如人的DNA鏈完整展開約2m長,經(jīng)過這樣的折疊就變成了納米級至微米級的染色質結構而可以儲存在小小的細胞核)。而DNA的復制轉錄是需要將DNA的緊密結構打開,從而允許一些調(diào)控因子結合(轉錄因子或其他調(diào)控因子)。這部分打開的染色質,就叫開放染色質,打開的染色質允許其他調(diào)控因子結合的特性稱為染色質的可及性(chromatin accessibility)。因此,認為染色質的可及性與轉錄調(diào)控密切相關。
開放染色質的研究方法有ATAC-seq以及傳統(tǒng)的DNase-Seq及FAIRE-seq等,ATAC-Seq由于所需細胞量少,實驗簡單,可以在全基因組范圍內(nèi)檢測染色質的開放狀態(tài),目前已經(jīng)成為研究染色質開放性的首選技術方法。

技術詳情
ATAC-Seq、ChIP-Seq、Dnase-Seq、MNase-Seq、FAIRE-Seq整體的分析思路一致,找到富集區(qū)域,對富集區(qū)域進行功能分析。
? ChIP-Seq是揭示特定轉錄因子或蛋白復合物的結合區(qū)域,實際是研究DNA和蛋白質的相互作用,利用抗體將蛋白質和DNA一起富集,并對富集到的DNA進行測序。
? DNase-Seq、ATAC-Seq、FAIRE-Seq都是用來研究開放染色質區(qū)域。DNase-Seq是用的DNase I內(nèi)切酶識別開放染色質區(qū)域,而ATAC-seq是用的Tn5轉座酶,隨后進行富集和擴增;FAIRE-Seq是先進行超聲裂解,然后用酚-氯仿富集。
? MNase-Seq是用來鑒定核小體區(qū)域。
圖片來源: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.[ ]
參考文獻
[[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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