Lee Heather J, Smallwood Sébastien A
Epigenetics Programme, Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK.
School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, The University of Newcastle, Callaghan, 2308, NSW, Australia.
Methods Mol Biol. 2018;1712:87-95. doi: 10.1007/978-1-4939-7514-3_7.
DNA methylation is an epigenetic mark implicated in the regulation of key biological processes. Using high-throughput sequencing technologies and bisulfite-based approaches, it is possible to obtain comprehensive genome-wide maps of the mammalian DNA methylation landscape with a single-nucleotide resolution and absolute quantification. However, these methods were only applicable to bulk populations of cells. Here, we present a protocol to perform whole-genome bisulfite sequencing on single cells (scBS-Seq) using a post-bisulfite adapter tagging approach. In this method, bisulfite treatment is performed prior to library generation in order to both convert unmethylated cytosines and fragment DNA to an appropriate size. Then DNA fragments are pre-amplified with concomitant integration of the sequencing adapters, and libraries are subsequently amplified and indexed by PCR. Using scBS-Seq we can accurately measure DNA methylation at up to 50% of individual CpG sites and 70% of CpG islands.
DNA甲基化是一种表观遗传标记，与关键生物学过程的调控有关。利用高通量测序技术和基于亚硫酸氢盐的方法，能够以单核苷酸分辨率和绝对定量获得哺乳动物DNA甲基化图谱的全基因组综合图谱。然而，这些方法仅适用于大量细胞群体。在此，我们展示了一种使用亚硫酸氢盐后衔接子标记方法对单细胞进行全基因组亚硫酸氢盐测序（scBS-Seq）的方案。在该方法中，亚硫酸氢盐处理在文库构建之前进行，以便将未甲基化的胞嘧啶转化并将DNA片段切割成合适大小。然后，DNA片段通过测序衔接子的伴随整合进行预扩增，随后文库通过PCR进行扩增和索引。使用scBS-Seq，我们能够准确测量高达50%的单个CpG位点和70%的CpG岛的DNA甲基化情况。
