m6A Dot Blot Quantification Protocol

m6A dot blotting was performed as previously described [49 (link)]. Briefly, isolated total RNA was measured using a NanoDrop instrument, and 200–400 ng RNA was spotted onto a Hybond-N + membrane (Invitrogen, USA). Then, membranes were crosslinked using UV irradiation and incubated with m6A-specific antibody (Abcam, 1:1000 dilution) overnight at 4 °C, after blocking with 5% nonfat milk in PBST. Then, diluted HRP-conjugated secondary antibody (1:10000) was added to the membrane for 1 h at room temperature. Membranes were developed using an ECL chromogenic kit (Beyotime, China) and the signal detected using a the Mini-REPORT Tetra Electrophoresis System. Methylene blue staining was used as a loading control.