m6A-RNA immunoprecipitation-qPCR (MeRIP-qPCR)

MeRIP-qPCR was performed as previously reported (31). In brief, mRNA was isolated, purified and chemically shredded into ~100-nt fragments using Ambion fragmentation reagent (Thermo Fisher Scientific, Inc.). mRNA fragments (200 ng) were denatured at 65°C for 5 min and incubated with 20 µl of Magna ChIP Protein A+G Magnetic Beads (2923270, Millipore) conjugated to 1 µg of anti-m⁶A polyclonal antibody (ab208577, Abcam) or mouse control IgG (sc-2025, Santa Cruz Biotechnology, Inc.) in 1X IPP buffer (15 mM NaCl, 10 mM Tris-HCl and 0.1% NP-40). mRNA was incubated with m⁶A-bound beads with rotation at 4°C for 3 h in IPP buffer. The beads were washed twice with 1X IPP buffer, twice with low-salt buffer (50 mM NaCl, 10 mM Tris-HCl and 0.1% NP-40), twice with high-salt buffer (500 mM NaCl, 10 mM Tris-HCl and 0.1% NP-40) and once with 1X IPP buffer. RNA was eluted from the beads with 50 µl of RLT buffer and purified through Qiagen RNeasy columns (Qiagen) according to the manufacturer's recommendation. Total RNA was finally eluted in 100 µl of RNase-free water. The relative abundances of RNAs of interest were measured using qPCR and normalized to the input level.