GSM7720086: RA1 shFTO ip; Homo sapiens; RIP-Seq
Total RNA was extracted using TRIzol™ Reagent (Invitrogen, 15596018). The concentration of total RNA was measured by Qubit RNA HS assay kit (Invitrogen, Q32852). 10μg total RNA was fragmented into 100-200 nt RNA fragments using 10X RNA Fragmentation Buffer (100 mM Tris-HCl, 100 mM ZnCl2 in nuclease-free H2O). The reaction was stopped by adding 10X EDTA (0.5M EDTA). Methylated RNA immunoprecipitation was performed using EpiTM m6A immunoprecipitation kit (Epibiotek, R1804). Briefly, the fragmented RNA was incubated with anti-m6A monoclonal antibody (abcam,ab208577) for 3 h at 4℃ and then with protein A/G magnetic beads (Invitrogen, 8880210002D/10004D) at 4 ℃ for an additional 2 h to obtain immunoprecipitated RNA fragments. The m6A-enriched RNA was purified using TRIzol™ Reagent (Invitrogen, 15596018). The library was prepared by smart-seq method. Both the input samples without IP and the m6A IP samples were subjected to 150-bp, paired-end sequencing on an Illumina NovaSeq 6000 sequencer. meRIP-seq