Enol chloroform isoamylol

Total RNAs were extracted with RNAiso Plus (TaKaRa). For meRIP, the procedure was described previously. In brief, purified mRNAs (5 µg) were digested by DNase I (M0303, NEB) and then fragmented into around 200 nt fragments by incubation at 95 °C for 25 s in RNA Fragmentation Reagents (Ambion, AM8740), followed by standard ethanol precipitation and collection. Anti-m⁶A antibody (10 µg antibody for 5 µg mRNAs; Synaptic Systems) was incubated with 40 µl Protein A beads (Sigma, P9424) in IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.4) for 2 h at room temperature. The fragmented mRNAs (5 µg) were incubated with the prepared antibody-bead mixture for 4 h at 4 °C. By washing three times, bound RNA was eluted from the beads with 0.5 mg/ml N6-methyladenosine (BERRY & ASSOCIATES, P3732) in IPP buffer. The eluted RNA was extracted by Enol:Chloroform:Isoamylol (pH < 5.0, Solarbio life science, P1025) and then generated to cDNA using 5 x All-In-One RT MasterMix (ABM, G490). The enrichment of m6A was quantified by qPCR. The sequences of qPCR primers are listed in Supplementary Table S2.