染色质免疫沉淀外切酶方法：以近碱基对精度鉴定蛋白质与DNA的相互作用。
The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision.
作者信息
Perreault Andrea A, Venters Bryan J
机构信息
Department of Molecular Physiology and Biophysics, Vanderbilt University.
Department of Molecular Physiology and Biophysics, Vanderbilt University;
出版信息
J Vis Exp. 2016 Dec 23(118):55016. doi: 10.3791/55016.
Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA interactions. ChIP coupled to high throughput sequencing (ChIP-seq) is commonly used to determine the genomic location of proteins that interact with chromatin. However, ChIP-seq is hampered by relatively low mapping resolution of several hundred base pairs and high background signal. The ChIP-exo method is a refined version of ChIP-seq that substantially improves upon both resolution and noise. The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5' DNA borders of the protein-DNA crosslink site. The ChIP-exo libraries are then subjected to high throughput sequencing. The resulting data can be leveraged to provide unique and ultra-high resolution insights into the functional organization of the genome. Here, we describe the ChIP-exo method that we have optimized and streamlined for mammalian systems and next-generation sequencing-by-synthesis platform.
染色质免疫沉淀（ChIP）是表观遗传学和基因调控领域中不可或缺的工具，用于分离特定的蛋白质 - DNA 相互作用。ChIP 与高通量测序相结合（ChIP-seq）常用于确定与染色质相互作用的蛋白质的基因组位置。然而，ChIP-seq 受到几百个碱基对的相对较低的映射分辨率和高背景信号的阻碍。ChIP-exo 方法是 ChIP-seq 的改进版本，在分辨率和噪声方面都有显著提高。ChIP-exo 方法的关键区别在于在文库制备工作流程中加入了λ核酸外切酶消化，以有效地确定蛋白质 - DNA 交联位点左右两侧 5' DNA 边界的足迹。然后对 ChIP-exo 文库进行高通量测序。所得数据可用于提供关于基因组功能组织的独特和超高分辨率见解。在这里，我们描述了我们针对哺乳动物系统和新一代合成测序平台优化和简化的 ChIP-exo 方法。
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