m6A MeRIP-Seq of interscapular brown adipose tissue (iBAT) in Mettl3flox/flox and BAT-specific Mettl3 knockout (BKO) mice

Purpose: Through mRNA m6A-profiling, we aim to characterize the mRNA m6A changes in iBAT obtained from Mettl3flox/flox and BKO mice. Methods: Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from iBAT of Mettl3 flox/flox and BKO mice at 8 weeks old. Each sample (300 µg total RNA) was pooled from 5 mice for each group. Poly(A)+ RNA was purified using Dynabeads™ mRNA Purification Kit (Invitrogen) following the manufacturer's instructions. Chemically fragmented poly(A)+ RNA was incubated with m6A antibody (Synaptic System, 202003) for immunoprecipitation following the standard protocol of Magna MeRIPTM m6A Kit (MERK, 17-10499). Enrichment of m6A mRNA was then analyzed by high-throughput sequencing using Illumina Hiseq X platform. The m6A peaks were detected by MACS2. The motif search was detected by HOMER. Conclusion: The iBAT mRNA m6A profiles in Mettl3flox/flox and BKO mice were characterized. Overall design: To map the mRNA m6A modification caused by METTL3 in iBAT, MeRIP-seq was performed in iBAT obtained from Mettl3flox/flox and BKO mice.