Montanera Kaitlin N, Rhee Ho Sung
Department of Cell and Systems Biology, University of Toronto; Department of Biology, University of Toronto.
Department of Cell and Systems Biology, University of Toronto; Department of Biology, University of Toronto;
J Vis Exp. 2020 Aug 14(162). doi: 10.3791/61124.
Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) is widely used to identify genome-wide binding locations of DNA-binding proteins. However, the ChIP-seq method is limited by its heterogeneity in length of sonicated DNA fragments and non-specific background DNA, resulting in low mapping resolution and uncertainty in DNA-binding sites. To overcome these limitations, the combination of ChIP with exonuclease digestion (ChIP-exo) utilizes 5' to 3' exonuclease digestion to trim the heterogeneously sized immunoprecipitated DNA to the protein-DNA crosslinking site. Exonuclease treatment also eliminates non-specific background DNA. The library-prepared and exonuclease-digested DNA can be sent for high-throughput sequencing. The ChIP-exo method allows for near base-pair mapping resolution with greater detection sensitivity and reduced background signal. An optimized ChIP-exo protocol for mammalian cells and next-generation sequencing is described below.
识别基因组上特定的蛋白质 - DNA 相互作用对于理解基因调控至关重要。染色质免疫沉淀结合高通量测序（ChIP-seq）被广泛用于识别 DNA 结合蛋白在全基因组范围内的结合位点。然而，ChIP-seq 方法受到超声破碎的 DNA 片段长度异质性和非特异性背景 DNA 的限制，导致映射分辨率低以及 DNA 结合位点的不确定性。为了克服这些限制，ChIP 与核酸外切酶消化相结合（ChIP-exo）利用 5' 到 3' 核酸外切酶消化将大小不均一的免疫沉淀 DNA 修剪到蛋白质 - DNA 交联位点。核酸外切酶处理还消除了非特异性背景 DNA。经文库制备和核酸外切酶消化的 DNA 可以送去进行高通量测序。ChIP-exo 方法能够实现近乎碱基对水平的映射分辨率，具有更高的检测灵敏度并降低背景信号。以下描述了一种针对哺乳动物细胞和下一代测序的优化 ChIP-exo 方案。
