HELP-tagging assay design and library preparation. The genomic DNA is digested by HpaII or MspI, the former only cutting at CCGG sequences where the central CG dinucleotide is unmethylated. The first Illumina adapter (AE) is ligated to the compatible cohesive end created, juxtaposing an EcoP15I site beside the HpaII/MspI digestion site and allowing EcoP15I to digest within the flanking DNA sequence as shown. An A overhang is created, allowing the ligation of the second Illumina adapter (AS, green). This will create not only AE-insert-AS products but also AS-insert-AS molecules. By performing a T7 polymerase-mediated in vitro transcription from a promoter sequence located on the AE adapter, we can selectively enrich for the AE-insert-AS product, following which limited PCR amplification is performed to generate a single sized product for Illumina sequencing. RT, reverse transcription.
Suzuki et al. Genome Biology 2010 11:R36 doi:10.1186/gb-2010-11-4-r36