1260 infinity 2

Detection of DNA methylation levels was adapted from a previously described protocol 16 (link). Cells were lysed in buffer (20 mM Tris-HCl pH 8.0, 10 mM NaCl, 10 mM EDTA pH 8.0, 0.5% SDS) at 37 °C overnight, then 100 μg/mL proteinase K was added and allowed to digest proteins at 60 °C overnight. DNA was extracted using phenol:chloroform:isoamyl alcohol 25:24:1 (Cat. #BSA03M1, BioFlux) and chloroform, precipitated in isopropanol at -20 °C for 30 min, then digested by nuclease P1 (Cat. #M0660S, NEB, UK) and dephosphorylated by antarctic phosphatase (Cat. #M289S, NEB, UK) at 37 °C for 4 h. 5-methyl-deoxycytosine and deoxycytosine were detected with 280 nm UV by an HPLC system (Agilent, 1260 Infinity II, USA) with a BEH C18 column (Cat. #186003625, Waters XBridge, 5 μm, 250 mm × 4.6 mm, USA). Separation was achieved at a flow rate of 0.5 mL/min using the following linear gradient: 0%-70% buffer B (100% MeOH) for 25 min, 70%-0% buffer B for 20 min. Buffer A was composed of 10 mM KH₂PO₄, pH 3.6. Nucleotides were analyzed by OpenLAB CDS ChemStation based on peaks areas.