Li Ren, Cui Xia
Key Laboratory of Biology and Genetic Improvement of Horticultural Crops of the Ministry of Agriculture, Sino-Dutch Joint Laboratory of Horticultural Genomics, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China.
Methods Mol Biol. 2018;1830:367-379. doi: 10.1007/978-1-4939-8657-6_22.
DNase I hypersensitive sites (DHSs) are genomic regions that exhibit hypersensitivity to DNase I cleavage. DHSs appear to be an essential feature of "open chromatin" structure in eukaryotes. Most of regulatory elements and the majority of transcription factor-binding sites are associated with open chromatin marked by DHSs. DNase I digestion followed by high-throughput DNA sequencing (DNase-seq) has become a powerful tool to reveal chromatin status and identify regulatory elements at genome-wide level. Here, we developed a DNase-seq procedure in tomato revised from previous methods in other plants, involving plant nuclei isolation, digestion of purified nuclei with DNase I, collection of DNase-trimmed DNA fragments, validation of DHS sample quality by qPCR, and DNase-seq library preparation. We introduced an AMPure XP beads system for the selection of the desirable DNA fragment. DHS datasets will provide the dynamic profiles of regulatory elements during plant development.
脱氧核糖核酸酶I超敏位点（DHSs）是对脱氧核糖核酸酶I切割表现出超敏性的基因组区域。DHSs似乎是真核生物中“开放染色质”结构的一个基本特征。大多数调控元件和大多数转录因子结合位点都与以DHSs为标志的开放染色质相关。脱氧核糖核酸酶I消化后进行高通量DNA测序（DNase-seq）已成为在全基因组水平上揭示染色质状态和识别调控元件的强大工具。在此，我们改进了一种在番茄中进行DNase-seq的方法，该方法是在其他植物先前方法的基础上修订而来的，包括植物细胞核分离、用脱氧核糖核酸酶I消化纯化的细胞核、收集经脱氧核糖核酸酶修剪的DNA片段、通过qPCR验证DHS样本质量以及制备DNase-seq文库。我们引入了一种AMPure XP磁珠系统来选择所需的DNA片段。DHS数据集将提供植物发育过程中调控元件的动态图谱。
