Anti 5fc antiserum

Chemical labeling of 76mer model DNA containing single 5fC or Tdg^−/− mESC gDNA was performed as described above. For the dot blot assay, different amounts of model DNA or gDNA were denatured in advance with NaOH solution (final concentration of 0.15M), spotted on the Amersham Hybond-N+ membrane (GE Healthcare) and air-dried for 5 min. The membrane was UV crosslinked and then blocked with 5% nonfat milk in 1x TBST at room temperature for 2 h. The membrane was then incubated with anti-5fC antiserum (Active Motif, 61223, 1:2,500 dilution) overnight at 4 °C followed by 1× TBST washing three times. After incubation with HRP-conjugated anti-rabbit IgG secondary antibody (CW Biotech, CW0103, 1:10,00 dilution) at room temperature for 1 h and washing with 1× TBST for three times, the membrane was supplied with 1 mL SuperSignal West Chemiluminescent Substrate (Thermo Scientific) and then visualized by chemiluminescence exposure.