Dot Blotting for DNA Methylation Analysis

Dot blotting was performed following the method described in Hainer et al.^{45 (link)}. Briefly, serial dilutions of genomic DNA starting at 300 ng were denatured at 95 °C for 10 min, then put on ice immediately for 10 min. Denatured DNA samples were spotted onto Amersham Hybond N + nylon membrane (GE Healthcare, Uppsala, Sweden) and the membranes were UV crosslinked. The membrane was incubated in 0.1% SDS overnight, blocked with 5% nonfat milk and 3% BSA for 2 h, incubated with anti-5mC (1:1000, Eurogentec BI-MECY, RRID:AB_2616058) for 1 h, washed three times with PBS-T, incubated with HRP conjugated anti-mouse secondary (1:10,000, Bio-Rad, Hercules, CA, USA, 170–6516, RRID:AB_11125547) for 1 h, washed three times with PBS-T, and detected with enhanced chemiluminescence. For loading, gDNA samples were diluted simultaneously, spotted directly onto Amersham Hybond N + nylon membrane (GE Healthcare) and the membranes were UV crosslinked. Membranes were incubated with 0.2% methylene blue for 5 min and washed five times with water.