MeRIP-seq of T3-12-03
Total RNA was extracted using the TRIzol reagent, and the concentration of total RNA was determined using a Qubit RNA HS assay kit. Total RNA (10 g) was fragmented into 100200 nt RNA fragments using 10 RNA fragmentation buffer (100 mM Tris-HCl, 100 mM ZnCl2, and nuclease-free H2O). The reaction was stopped by the addition of 10EDTA (0.5 M EDTA). Methylated RNA was immunoprecipitated using an EpiTM m6A Immunoprecipitation Kit. The fragmented RNA was incubated with an anti-m6A monoclonal antibody for 3 h at 4C and then incubated with Protein A/G magnetic beads for another 2 h at 4C to obtain immunoprecipitated RNA fragments. The m6A-rich RNA was purified using TRIzol reagent. Libraries were prepared using the SMART sequence method. Input samples without IP and m6A IP were sequenced at 150-bp paired-end on an Illumina NovaSeq 6000 sequencer.