Genomic DNA was isolated from biological replicates of frozen non-diseased human cardiac tissues (n=29)  using the QIAGEN DNeasy Blood & Tissue Kit according to the instructions provided by the manufacturer Typically, 1 µg of DNA was used to make an reduced representation bisulfite sequencing  (RRBS) library.  Genomic DNA was digested with methyl sensitive MspI enzyme which cut at every CCGG regardless of methylation status. Following enzymatic digestion, DNA end repiar and A-tailing were conducted by adding Klenow fragment and dNTP mixture. Next, DNA was then ligated to indexed TrueSeq adapters (Illumina) according to manufacturer's protocol. Adapted ligated DNA fragments were then size selected for the desired input fragments (150-250bps) and (250-350bps) with AMPure XP beads (Beckman Coulter). Bisulfite conversion of size selected libraries was then performed by using Epimark Bisulfite Conversion kit (New Englands Biolabs) using manufacturer's instructions. Final libraries from RRBS were generated by PCR amplification and pooled libraries were quantified using Qubit fluorometer. DNA libraries were then sequenced on Illumina NextSeq 500 machine generating 75 bp single end reads and base calling was performed  using standard Ilumina base calling software.
GEO Accession
GSM4376937