Methylated RNA Immunoprecipitation (meRIP) and RT-qPCR

3 × 10⁷ Hela or 4 × 10⁷ Siha cells were prepared for RNA extraction. Total RNAs were extracted by using Trizol (Sigma, USA). mRNA was extracted by using PolyATtract^® Systems IV (Promega, USA) kit. The co-precipitated RNA was obtained according to the standard protocol of the Magna MeRIP m⁶A Kit (Merck Millipore, Germany). Briefly, 50 μl protein A/G beads were incubated with 2.5 μg anti-m⁶A antibody for 30 min at room temperature. Then 5 μg fragmented mRNA was incubated with anti-m⁶A antibody coated protein A/G beads at 4°C overnight immunoprecitation. After washes, RNA isolation and RT-qPCR were performed as described previously.