5prime phase lock gel heavy

Trypsinized ESC were lysed for 2 to 3 hours at 55°C with lysis buffer [100 mM tris-HCl (pH 8), 5 mM EDTA, 0.2% SDS, 20 mM NaCl, and Proteinase K (100 μg/ml)]. DNA was isolated by adding an equal volume of phenol/chloroform/isoamyl to the samples and using phase-lock tubes (5PRIME Phase Lock Gel Heavy, Quantabio), followed by an extraction with identical volume of chloroform. DNA was precipitated with two volumes of 100% ethanol plus 0.3 M NaAc, washed with 70% ethanol, and resuspended in water. A total of 500 ng of DNA were diluted in 0.3 M NaOH and denatured at 42°C for 12 min. After incubation, the samples were rapidly transferred by spotting each sample into a nitrocellulose membrane (nitrocellulose blotting membrane, GE Healthcare). After the transfer, DNA was cross-linked with a Stratalinker ultraviolet cross-linker (Stratagene) using the autocrosslink setting. The membrane was blocked in 5% skim milk (Millipore) and 0.1% Tween 20 (Sigma-Aldrich) in PBS, incubated with 1:500 dilution of the anti-5mC antibody (see table S2) overnight at 4°C, followed by incubation with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature. Membrane was developed using SuperSignal West Pico PLUS (Thermo Fisher Scientific). See table S2 for antibody information.