Identification of Open Chromatin Regions in Plant Genomes Using ATAC-Seq.
Identification of Open Chromatin Regions in Plant Genomes Using ATAC-Seq.
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DOI：
10.1007/978-1-4939-7318-7_12
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发表时间：
2018
期刊：
影响因子：
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通讯作者：
Deal RB
中科院分区：
文献类型：
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作者：
Bajic M;Maher KA;Deal RB
识别和表征高度可及的染色质区域有助于确定基因组调控元件的位置和理解转录调控。在本章中，我们描述了一种利用转座酶可及染色质分析结合高通量测序(ATAC-SEQ)来定位植物可及染色质特征的方法，ATAC-SEQ最初是为培养的动物细胞开发的。该技术利用高活性的Tn5转座酶来引起DNA切割，并同时将测序适配子插入到输入核的开放染色质区域。ATAC-SEQ在植物组织中的应用一直是具有挑战性的，因为很难分离出足够不受干扰的细胞器DNA的核。在这里，我们提出了两种不同的方法来纯化ATAC-SEQ植物的细胞核：完整的方法(在特定细胞类型中标记的核的分离)从植物的单个细胞类型中分离出核，以及组织裂解和蔗糖沉淀法以分离出足够纯度的总核。我们提供了使用这两种方法分离的核的转座酶处理的详细说明，以及随后ATAC-seq文库的准备。测序就绪的ATAC-seq文库可以在短短一天内从植物组织中制备出来。这里描述的程序是针对拟南芥进行优化的，但也可以应用于其他植物物种。
Identifying and characterizing highly accessible chromatin regions assists in determining the location of genomic regulatory elements and understanding transcriptional regulation. In this chapter we describe an approach to map accessible chromatin features in plants using the Assay for Transposase Accessible Chromatin, combined with high throughput sequencing (ATAC-seq), which was originally developed for cultured animal cells. This technique utilizes a hyperactive Tn5 transposase to cause DNA cleavage and simultaneous insertion of sequencing adapters into open chromatin regions of the input nuclei. The application of ATAC-seq to plant tissue has been challenging due to the difficulty of isolating nuclei sufficiently free of interfering organellar DNA. Here we present two different approaches to purify plant nuclei for ATAC-seq: the INTACT method (Isolation of Nuclei TAgged in Specific Cell Types) to isolate nuclei from individual cell types of the plant, and tissue lysis followed by sucrose sedimentation to isolate sufficiently pure total nuclei. We provide detailed instructions for transposase treatment of nuclei isolated using either approach, as well as subsequent preparation of ATAC-seq libraries. Sequencing-ready ATAC-seq libraries can be prepared from plant tissue in as little as one day. The procedures described here are optimized for Arabidopsis thaliana but can also be applied to other plant species.