4.9. M6A dot blot assay

The cells were lysed with Qiagen reagent or Trizol reagent, and total RNA was extracted. After the sufficient mix of 5 μl RNA samples, 15 μl formaldehyde/SSC buffer (10× SSC contains 6.15 mol/L formaldehyde) (Sigma‐Aldrich, Saint Louis, MO, USA) and RNA incubation buffer, draw a circle on the NC membrane with a hydrophobic pen to prevent droplet dispersion, then spot 5–8 μl of pre‐treated RNA sample on the NC membrane. Upon drying, the RNA on the membrane was cross‐linked in ultraviolet cross linker. The crosslinked membrane was incubated with 0.02% methylene blue (Sigma‐Aldrich) for 5–10 min. Upon washing 5 min with TBST, the stained membrane was photographed and then incubated with the antibody of anti‐m6A (Cell Signaling Technology, 56593) overnight at 4℃. Then the second antibody was incubated for 2.5 h. The ECF developer was diluted with TBS in proper proportion, and the membrane was scanned on the GE Healthcare.