Application of Microdroplet PCR for Large-Scale Targeted Bisulfite Sequencing. Homo sapiens

Cytosine methylation of DNA CpG dinucleotides in gene promoters is an epigenetic modification that regulates gene transcription. While many methods exist to interrogate methylation states, no current methods offer large-scale, targeted, single CpG resolution. We report an approach combining bisulfite treatment followed by RainDance microdroplet PCR with next-generation sequencing to assay the methylation state of 50 genes in the regions 1 kb upstream and downstream of their transcription start sites. Overall design: Wildtype and hypermethylated Jurkat DNA (New Englad Biolabs) was treated with bisulfite to convert all unmethylated cytosines to uracil. Following bisulfite treatment, targeted amplification was carried out using a custom primer library and microdroplet PCR. PCR product was sheared to 200 bp and ligated to sequencing adapters following standard protocols. Sequencing was conducted with single-end 100 bp reads on an Illumina GAIIx for wild type Jurkat DNA or Jurkat CpG DNA with a single sample per lane.