Dot blotting for DNA 6mA

Dot blotting was performed as described previously with minor modifications (Zhang et al., 2015). In brief, the synthesized oligos (Wu et al., 2016) were loaded on an HATF00010 nitrocellulose membrane (Merck Millipore) and air dried for 5 min. The membrane was baked at 80°C for 2 h and then blocked in blocking buffer (5% milk in PBST) for 2 h at room temperature. The membrane was incubated with a specific anti-6mA antibody (Synaptic systems; 1:2000) overnight at 4°C, then incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody {"type":"entrez-nucleotide","attrs":{"text":"M21003","term_id":"175065","term_text":"M21003"}}M21003 (Abmart; 1:2000) at room temperature for 1.5 h. The antibody-bound 6mA was then incubated with a high-sensitivity enhanced chemiluminescence reagent (Sangon Biotech) and detected and quantified with a FUSION Solo 6S imaging system (VILBER).