Anti 5mc antibody

Cells were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100, followed by blocking with 10% FBS in PBS for 1 h. Samples were stained with primary antibodies for OCT4 (Santa Cruz Biotechnology, sc-5279), NANOG (R&D Systems, AF1997), SOX2 (Millipore, AB5603), PAX6 (Santa Cruz Biotechnology, sc-81649), and GATA6 (Cell Signaling Technology, 5851) overnight at 4 °C. Secondary antibody staining was performed for 1 h at room temperature with Alexa Fluor 488-donkey anti-goat IgG, Alexa Fluor 555-donkey anti-mouse IgG, and Alexa Fluor 555-donkey anti-rabbit IgG (ThermoFisher Scientific). For 5-hmC staining, cells were treated with 1.5 M HCl for 30 min at room temperature after 4% paraformaldehyde fixation (antibody: Active Motif, 39769). For 5-mC detection, cells were fixed with ice-cold 70% ethanol for 5 min, followed by 1.5 M HCl for 30 min at room temperature. Samples were blocked with 5% FBS and 0.3% Triton X-100 in PBS for 1 h and then stained with anti-5-mC antibody (Cell Signaling Technology, 28692) diluted in PBS with 1% BSA and 0.3% Triton X-100 overnight at 4 °C. Secondary antibody staining was performed for 1 h at room temperature with Alexa Fluor 555-donkey anti-rabbit IgG (ThermoFisher Scientific). All images were taken using Olympus IX71 fluorescence microscope.

MeDIP was performed as described previously [39 (link)]. Genomic DNA was sonicated for 30 min (30 s ON, 30 s OFF) using Bioruptor Plus (Diagenode), and each DNA sample was incubated with 5 µl of anti-5-mC antibody (Cell signaling) overnight at 4 °C. The samples were then incubated with 30 µl of Protein G magnetic beads (Thermo Scientific) for 2 h at 4 °C. After eluted from the beads, the samples were incubated with proteinase K for 2 h at 65 °C, and purified DNA was analyzed for methylated DNA enrichment at the promoter region of GRIA1 [36 (link)], detected by qPCR using the SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara). MeDIP Ct values were normalized against 2% input.