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Low Input Whole-Genome Bisulfite Sequencing Using a Post-Bisulfite Adapter Tagging Approach.

Methods in molecular biology (Clifton, N.J.) (2017-12-11)
Julian R Peat, Sébastien A Smallwood
ABSTRACT

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.

MATERIALS
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Sigma-Aldrich
Imprint® DNA Modification Kit, For bisulfite DNA conversion & purification