Unmethylated control genomic DNA was obtained from either DNA extracted from DNMT1/DNMT3b double-knockout (DKO) cells (39 (link)), human male genomic DNA (Promega) or Epitect unmethylated control DNA (Qiagen). Enzymatically CpG-methylated HeLa genomic DNA (New England BioLabs) was used as a fully-methylated control.
All genomic DNA was processed according to the ‘Methylation-on-Beads’ (MOB) bisulfite conversion technique as previously published (40 (link)). Briefly, for samples requiring extraction, cells or plasma were first digested in a solution containing 3 ml of Buffer AL (Qiagen) and 1 ml of Proteinase K (Invitrogen). DNA was then extracted via precipitation by isopropanol, then purified and washed by a series of magnetic decantation steps. The resulting DNA was bisulfite converted using reagents contained within the EZ DNA Methylation Kit (Zymo Research) according to the MOB protocol, washed by magnetic decantation and eluted into a final volume of 100 μl.
All genomic DNA was processed according to the ‘Methylation-on-Beads’ (MOB) bisulfite conversion technique as previously published (40 (link)). Briefly, for samples requiring extraction, cells or plasma were first digested in a solution containing 3 ml of Buffer AL (Qiagen) and 1 ml of Proteinase K (Invitrogen). DNA was then extracted via precipitation by isopropanol, then purified and washed by a series of magnetic decantation steps. The resulting DNA was bisulfite converted using reagents contained within the EZ DNA Methylation Kit (Zymo Research) according to the MOB protocol, washed by magnetic decantation and eluted into a final volume of 100 μl.
