HELP-tagging of homo sapiens: pediatric CD4+ T-cells: Sample A42_m
1 μg of Th cell genomic DNA, digested by HpaII, was purified and ligated at the cohesive end to the first adaptor including a restriction enzyme site that was then digested by EcoP15I followed by ligation of the second adaptor that was Illumina sequencer compatible. These adaptors served as priming sites for ligation-mediated PCR amplification. To generate longer flanking sequences, a T7 polymerase was added and followed by a reverse transcription step. The libraries were sequenced using Illumina HiSeq 2500 at the Einstein Epigenomics Core facility and compared with a reference human MspI library. While MspI digestion is methylation insensitive and cuts the DNA at all CCGG sites, HpaII digestion is methylation sensitive, and cuts DNA only at CCGG sites where the central CG is unmethylated at the cytosine nucleotide. Comparison of HpaII count to MspI count for each locus thereby allows quantification of DNA  methylation. The angle obtained by plotting HpaII count on the y-axis and MspI count on the x-axis provides a quantitative measure of locus-specific methylation. A larger angle is formed when HpaII count is high relative to MspI and is evidence of relative hypomethylation while a smaller angle is evidence of hypermethylation.
NHLBI-phs001812
A42_HELPtagging