GSM5221348: merip_kd_3d_rep2; Schmidtea mediterranea; RIP-Seq
For m6A immunoprecipitation, the procedure was modified from the previously reported methods. In brief, purified mRNAs from control and mettl3 morphants were digested by DNase I and then fragmented into around 100-nt fragments by incubation at 94 °C for 1 min in fragmentation buffer (10 mM ZnCl2, 10 mM Tris-HCl, pH 7.0). The reaction was then stopped with 0.05 M EDTA (Ambion, AM8740), followed by standard ethanol precipitation and collection. Anti-m6A polyclonal antibody (12 μg antibody for 6 μg mRNAs; Synaptic Systems, 202003) was incubated with 50 μl Protein A beads (Sigma, P9424) in IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.4) for 1 h at room temperature. Then the mRNAs (6 μg) were incubated with the prepared antibody-beads mixture for 4 h at 4 °C. After washing, bound RNAs were extracted by TRNzol. Fragmented mRNA was used for library construction using the KAPA Stranded mRNA-Seq Kit (KAPA, K8401) according to the manufacturer's protocol.