Quantifying 5mC and 5hmC in Genomic DNA

The genomic DNA dot-blot analysis of 5mC and 5hmC was performed following the DNA Dot Blot Protocol (Cell Signaling, #28692), as previously described.^{52 (link)} Briefly, genomic DNA was extracted using Quick-DNA Miniprep Plus Kit (Zymo Research, D4068), and DNA concentration was measured by NanoDrop. The same amount of DNA was denatured with 10X DNA denaturing buffer (1 M NaOH and 0.1 M EDTA) and incubated at 95°C for 10 min, which was then immediately mixed with an equal volume of 20X SSC buffer, pH 7.0 (Invitrogen, 15557044) and chilled on ice. The DNA samples were diluted with a pre-determined amount and loaded on the positive-charged Nylon membrane (GE Amersham, RPN2020B) using a vacuum chamber (Manifold, SRC-96). The membrane was dried, auto-crosslinked with 1200 x100 μJ/cm², and blocked with 5% milk/TBST for 1 h. Next, the membrane was incubated with 5mC (Cell Signaling, 28692) or 5hmC (Active Motif, 39769) antibodies, the same as the western blot analysis.