m6A quantification using ELISA and dot blot

The m⁶A RNA methylation quantification kit (ab185912; Abcam, Cambridge, United Kingdom) was used to measure the m⁶A content of total RNA, following the manufacturer’s instructions. Positive and negative controls provided with the kit were used to compute m⁶A levels, according to the manufacturer’s instructions. This assessment was complemented by m⁶A dot blot assay. Briefly, 2000 ng of RNA were denatured at 95 °C for 3 min and then pipetted into nitrocellulose membranes. Membranes were dryed for 30 min at 37 °C following UV light exposure (125mJoules/cm²) for allowing crosslink between RNA and the membrane. Then, membranes were washed in TBS-0.1% Tween, blocked in 5% dry milk and incubated with primary antibody for m⁶A (Abcam, 1:1000) at 4 °C, overnight. After that, membranes were incubated with anti-rabbit secondary horseradish peroxidase conjugated antibody (1:5000, Cell Signaling Technology) for 1 h at room temperature and the signal was detected by chemiluminescence. Methylene blue staining solution (0.02% methylene blue in 0.3 M sodium acetate, pH 5.5) was used as loading control.