GSM5854047: TM_1_H; Ara; Bisulfite-Seq
Reduced representation bisulfite sequencing (RRBS) was performed as described in Klughammer et al., Cell Reports 2015, using 100 ng of genomic DNA for most samples, while occasionally going down to 1 ng for samples with low DNA amounts (Supplementary Table 1). To assess the bisulfite conversion efficiency independent of CpG context, methylated and unmethylated spike-in controls were added at a concentration of 0.1%. For most samples, DNA was digested using the restriction enzymes MspI and TaqI in combination (as opposed to only MspI in the original protocol) in order to increase genome-wide coverage. For certain older samples, only MspI was used Restriction enzyme digestion was followed by fragment end repair, A-tailing, and adapter ligation. Fi-nally, the libraries were size selected by performing a 0.75× cleanup with AMPure XP beads (Beckman Coulter, A63881) retaining fragments of about 100 bp to 1000 bp length. The amount of effective li-brary was determined by qPCR, and samples were multiplexed in pools of 10 with similar qPCR Ct values. The pools were then subjected to bisulfite conversion, followed by library enrichment with PCR. Enrichment cycles were determined using qPCR and ranged from 6 to 18 (median: 11). After con-firming adequate fragment size distributions on Bioanalyzer High Sensitivity DNA chips (Agilent),