RIP-qRT-PCR Assay for m6A Readers

RIP assays were performed essentially as described in our previously published study [24 (link), 25 (link)]. In brief, cells were lysed using polysome lysis buffer (5 mM HEPES (pH 7.4), 85 mM KCl, 1 mM DTT, 5 mM PMSF, 0.5% NP40, supplemented with RNase inhibitors (Invitrogen, USA) and PIC (protease inhibitors cocktail, Roche, Switzerland)) on ice for 10 min. After centrifugation, the supernatant was collected with 10% of the lysate serving as “input”. The remainder of the lysate was incubated with 50 μl of protein A/G magnetic beads (Life Technologies, USA) coupled with 2 μg of primary antibodies rotated overnight at 4 °C with IgG antibody as the control. RNA was isolated using TRIzol (Invitrogen, USA) and reverse-transcribed into cDNA for qRT-PCR detection using a Takara SYBR green kit (Takara, Japan). Primary antibodies against YTHDF1 (Abcam, ab220162), YTHDF2 (Abcam, ab220163), YTHDF3 (Abcam, ab220161), and N6-methyladenosine (m6A) (Abcam, ab220161) were used.