MeRIP/m6A-seq of human B cell
Total RNAs were extracted using TRIzol Reagent (InvitrogenTM). The concentrations of total RNAs were measured by a Qubit RNA HS assay kit (InvitrogenTM). 10g of total RNAs were 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 (0.5 M EDTA). Methylated RNA immunoprecipitation was performed using an EpiTM m6A immunoprecipitation kit (Epibiotek). Briefly, the fragmented RNAs were incubated with anti-m6A monoclonal antibody (Abcam) for 3 h at 4C and then with protein A/G magnetic beads (InvitrogenTM) at 4C for an additional 2 h to obtain immunoprecipitated RNA fragments. The m6A-enriched RNA was purified using TRIzol Reagent (InvitrogenTM). 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.