MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing.
MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing.
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DOI：
10.1038/nprot.2014.114
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发表时间：
2015-03
期刊：
影响因子：
14.8
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中科院分区：
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目前的高通量DNA测序技术能够获取数十亿个数据点，通过这些数据点可以查询无数的生物过程，包括遗传变异、染色质结构、基因表达模式、小RNA和蛋白质-DNA相互作用。在这里，我们描述了甲基C测序(MethylC-seq)文库制备方法，这是一种能够在单碱基分辨率下对全基因组范围内的胞嘧啶DNA甲基化状态进行鉴定的二维协议。这项技术包括基因组DNA的片段，然后是接头连接、亚硫酸氢盐转化和使用接头特异的PCR引物进行有限扩增，为测序做准备。到目前为止，该方法已经成功地应用于从原代细胞培养、分离的细胞和1000多个动植物样本的新鲜组织中分离出的基因组DNA。
Current high-throughput DNA sequencing technologies enable acquisition of billions of data points through which myriad biological processes can be interrogated, including genetic variation, chromatin structure, gene expression patterns, small RNAs and protein–DNA interactions. Here we describe the MethylC-sequencing (MethylC-seq) library preparation method, a 2-d protocol that enables the genome-wide identification of cytosine DNA methylation states at single-base resolution. The technique involves fragmentation of genomic DNA followed by adapter ligation, bisulfite conversion and limited amplification using adapter-specific PCR primers in preparation for sequencing. To date, this protocol has been successfully applied to genomic DNA isolated from primary cell culture, sorted cells and fresh tissue from over a thousand plant and animal samples.