mRNA decapping assay

C666 cells were plated into 6‐well plates for 24 h and transfected with YTHDF1‐specific or control siRNAs. After 24 h, RNA was extracted from the transfected cells, and the presence of the 5′‐cap was determined as described previously (Du et al, 2016). Briefly, the cytoplasmic RNA samples (3 µg) were treated with three units of 5′‐phosphate‐dependent exonuclease XRN1 (New England BioLabs, USA) for 2 h at 37°C according to the manufacturer’s protocol. The treated RNAs were then purified overnight by ethanol precipitation and subjected to qPCR along with the total RNA sample. Finally, the ratios of capped RNA were calculated by dividing the RNA expression in XRN1‐treated samples by that in total RNA samples.