GSM8291335: GSCs, shMDH2_03_in01; Homo sapiens; RIP-Seq
Total RNA was extracted using TRIzol reagent (Invitrogen) and treated with RNase-free DNase I (Roche) to deplete DNA contamination. PolyA RNA was purified using a GenElute mRNA Miniprep Kit (Sigma-Aldrich) as per the manufacturer's instructions and fragmented using an RNA fragmentation kit (Ambion). Note that 200 μg of fragmented RNA was incubated with 3 μg anti-m6A (Synaptic Systems, Cat# 202 003) in RIP buffer (150 mmol/L NaCl, 10 mmol/L Tris, and 0.1% NP-40) for 2 hours at 4°C, followed by addition of washed protein A/G magnetic beads (Millipore) and incubation at 4°C for a further 2 hours. Beads were washed 6 times in RIP buffer and incubated with 50 μL immunoprecipitation buffer containing 0.5 mg/mL m6AMP (Sigma-Aldrich) to elute RNA. Immunoprecipitated RNA was extracted with phenol/chloroform, and RNA samples were sent for next-generation sequencing.