GSM4560323: P493-6_Input_Tet_0h; Homo sapiens; RIP-Seq
Total RNAs were extracted with TRNzol (Invitrogen, 15596-018), then purified with the Dynabeads® mRNA purification kit (Ambion, 61006) for two rounds. To immunoprecipitated m6A, purified mRNAs were digested by DNase I and then incubated at 94 °C for 60 seconds in fragmentation buffer (10 mM ZnCl2, 10 mM Tris-HCl, pH 7.0), to cut them into pieces of ~ 100-nt. The reaction was stopped with 0.05 M EDTA (Ambion, AM8740), and was followed by standard ethanol precipitation and collection. Anti-m6A polyclonal antibody (12 ug antibody for 6 ug mRNAs; Synaptic Systems, 202003) was incubated with 50 ul Protein A beads (Sigma, P9424) in IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.4) for 1 hr at room temperature. The mRNAs (6 ug) were incubated with the prepared antibody-beads mixture for 4 hrs at 4 °C. After washing, bound RNAs were extracted by TRNzol. For the RNA-seq, fragmented RNAs were re-suspended in H2O and used for library generation with mRNA sequencing kit (Illumina).
GEO Accession
GSM4560323