Cord blood CD4 T cells - Methyl Seq
200 ng genomic DNA was isolated from resting 2-5x105 CD4+ T cells (n= 6 lean, 4 obese) using Quick-gDNA MiniPrep (Zymo Research, Irvine CA), sheared to 100-200 bp using Covaris Ultrasonicator (Covaris, Woburn MA) and verified using Agilent 2100 Bioanalyzer. DNA cytosine methylation levels in purified resting UCB CD4 T cells were measured at single base resolution using a targeted bisulfite sequencing approach (SureSelectXT Human Methyl-Seq enrichment system, Agilent, Santa Clara CA), focusing on regions where methylation is known to impact gene regulation (cancer tissue-specific differentially methylated regions or DMRs, GENCODE promoters, CpG islands, shores and shelves 4kb, DNase I hypersensitive sites and RefGenes). The ends of sheared DNA were repaired, 3 adenylated, and ligated with methylated adapters. These DNA fragments were then hybridized with 120 nt biotinylated RNA library fragments that recognize methylated DNA regions and isolated using streptavidin beads followed by bisulfite conversion using Zymo EZ DNA Methylation-Gold Kit (Zymo, Irvine CA), which converts unmethylated cytosines to uracils. Libraries were then PCR amplified, ligated with unique indices, and sequenced on Illumina NextSeq500 platform to generate 60 million 50 bp paired-end reads per sample. Efficiency of bisulfite conversion was measured using 20 pg of unmethylated phage lambda DNA spiked in with each sample before DNA fragmentation. The bisulfite non-conversion rate was calculated as the percentage of cytosines sequenced at cytosine reference positions in lambda genome.