Low Input Whole-Genome Bisulfite Sequencing Using a Post-Bisulfite Adapter Tagging Approach
Low Input Whole-Genome Bisulfite Sequencing Using a Post-Bisulfite Adapter Tagging Approach
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DOI：
10.1007/978-1-4939-7481-8_9
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发表时间：
2018-01-01
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影响因子：
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通讯作者：
Smallwood, Sebastien A.
中科院分区：
文献类型：
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作者：
Peat, Julian R.;Smallwood, Sebastien A.
表观遗传标记 5-甲基胞嘧啶赋予基因表达可遗传的调节作用，可以在细胞命运转变过程中动态调节。随着高通量测序技术的发展，现在可以获得哺乳动物 DNA 甲基化图谱的全面全基因组图谱，但将这些技术应用于有限的材料仍然具有挑战性。在这里，我们提出了一种优化的方案，使用亚硫酸氢盐后接头标记方法对低输入（100-5000 个体细胞）进行全基因组亚硫酸氢盐测序。在此策略中，在文库生成之前进行亚硫酸氢盐处理，以便将未甲基化的胞嘧啶和 DNA 片段转化为适当的大小。然后使用随机四聚体引发通过互补链合成添加测序接头，随后通过 PCR 扩增文库。
The epigenetic mark 5-methylcytosine confers heritable regulation of gene expression that can be dynamically modulated during transitions in cell fate. With the development of high-throughput sequencing technologies, it is now possible to obtain comprehensive genome-wide maps of the mammalian DNA methylation landscape, but the application of these techniques to limited material remains challenging. Here, we present an optimized protocol to perform whole-genome bisulfite sequencing on low inputs (100-5000 somatic cells) using a post-bisulfite adapter tagging approach. In this strategy, bisulfite treatment is performed prior to library generation in order to both convert unmethylated cytosines and fragment DNA to an appropriate size. Then sequencing adapters are added by complementary strand synthesis using random tetramer priming, and libraries are subsequently amplified by PCR.