Cho Jin Sun, Blitz Ira L, Cho Ken W Y
Department of Developmental and Cell Biology, School of Biological Sciences, University of California, Irvine, California 92697.
Department of Developmental and Cell Biology, School of Biological Sciences, University of California, Irvine, California 92697
Cold Spring Harb Protoc. 2019 Apr 1;2019(4):pdb.prot098335. doi: 10.1101/pdb.prot098335.
Transcriptional regulatory elements are typically found in relatively nucleosome-free genomic regions, often referred to as "open chromatin." Deoxyribonuclease I (DNase I) can digest nucleosome-depleted DNA (presumably bound by transcription factors), but DNA in nucleosomes or higher-order chromatin fibers is less accessible to the nuclease. The DNase-seq method uses high-throughput sequencing to permit the interrogation of DNase hypersensitive sites (DHSs) across the entire genome and does not require prior knowledge of histone modifications, transcription factor binding sites, or high quality antibodies to identify potentially active regions of chromatin. Here, discontinuous iodixanol gradients are used as a gentle preparation of the nuclei from embryos. Short DNase I digestion times are followed by size selection of digested genomic DNA, yielding DHS fragments. These DNA fragments are subjected to real-time quantitative polymerase chain reaction (qPCR) and sequencing library construction. A library generation method and pipeline for analyzing DNase-seq data are also described.
转录调控元件通常位于相对无核小体的基因组区域，常被称为“开放染色质”。脱氧核糖核酸酶I（DNase I）可以消化无核小体的DNA（推测被转录因子结合），但核小体或高阶染色质纤维中的DNA对该核酸酶的可及性较低。DNase-seq方法利用高通量测序来检测全基因组中的DNase超敏位点（DHS），并且不需要预先了解组蛋白修饰、转录因子结合位点或高质量抗体来识别潜在的染色质活性区域。在这里，不连续的碘克沙醇梯度被用于从胚胎中温和制备细胞核。短时间的DNase I消化后，对消化后的基因组DNA进行大小选择，产生DHS片段。这些DNA片段进行实时定量聚合酶链反应（qPCR）和测序文库构建。还描述了一种用于分析DNase-seq数据的文库生成方法和流程。
