Zhang Wenli, Jiang Jiming
Department of Horticulture, University of Wisconsin-Madison, 1575 Linden Drive, Madison, WI, 53706-1580, USA.
Methods Mol Biol. 2015;1284:71-89. doi: 10.1007/978-1-4939-2444-8_4.
Genomic regions associated with regulatory proteins are known to be highly sensitive to DNase I digestion and are termed DNase I hypersensitive sites (DHSs). DHSs can be identified by DNase I digestion followed by high-throughput DNA sequencing (DNase-seq). DNase-seq has become a powerful technique for genome-wide mapping of chromatin accessibility in eukaryotes with a sequenced genome. We have developed a DNase-seq procedure in plants. This procedure was adapted from the protocol originally developed for mammalian cell lines. It includes plant nuclei isolation, digestion of purified nuclei with DNase I, recovery of DNase-trimmed DNA fragments, DNase-seq library development, Illumina sequencing and data analysis. We also introduce a barcoding system for library preparation. We have conducted DNase-seq in both Arabidopsis thaliana and rice, and developed genome-wide open chromatin maps in both species. These DHS datasets have been used to detect footprints from regulatory protein binding and to reveal genome-wide nucleosome positioning patterns.
已知与调控蛋白相关的基因组区域对DNA酶I消化高度敏感，被称为DNA酶I超敏感位点（DHSs）。DHSs可通过DNA酶I消化后进行高通量DNA测序（DNA酶测序）来鉴定。DNA酶测序已成为在具有测序基因组的真核生物中进行全基因组染色质可及性图谱绘制的强大技术。我们在植物中开发了一种DNA酶测序程序。该程序改编自最初为哺乳动物细胞系开发的方案。它包括植物细胞核分离、用DNA酶I消化纯化的细胞核、回收经DNA酶修剪的DNA片段、DNA酶测序文库构建、Illumina测序和数据分析。我们还引入了一种用于文库制备的条形码系统。我们已在拟南芥和水稻中进行了DNA酶测序，并在这两个物种中绘制了全基因组开放染色质图谱。这些DHS数据集已用于检测调控蛋白结合的足迹，并揭示全基因组核小体定位模式。
