GSM4844997: B6_M_B_2 [OxBS-seq]; Mus musculus; Bisulfite-Seq
AllPrep DNA/RNA Mini Kit (Qiagen) was used to perform simultaneous isolation of RNA and DNA from 10^6 sorted naïve CD4+ T cells and resting B cells. Triplicate 1µg samples of genomic DNA isolated from either naive CD4+ T cells or resting B cells were fragmented using a Covaris sonication system (Covaris S2). Following fragmentation, the DNA samples were spiked with synthetic fragments of DNA pre-labelled by PCR with 5mC and 5hmC used in the analysis step to determine the bisulfite conversion efficiency of cytosine forms (unmodified, 5mC and 5hmC). Libraries were prepared using the TruSeq DNA library preparation kit v2 (Illumina, FC-121-2001) according to manufacturer's instructions with some modifications. After ligation of TruSeq methylated adapters (ME-100-0010, Illumina) and AMPure® XP (Beckman) bead purification half of the sample was oxidized by treatment with KRuO4 (15 mM in 0.05 M NaOH) for 1 hr, performed as previously described