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

Purpose: Through mRNA m6A-profiling, we aim to characterize the mRNA m6A changes in iBAT obtained from Wtapflox/flox and Wtap-BKO mice. Methods: Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from iBAT of Wtap flox/flox and Wtap-BKO mice at 8 weeks old. Each sample (300 μg total RNA) was pooled from 8 mice for each group. Poly(A)+ RNA was purified using Dynabeads™ mRNA Purification Kit (Invitrogen) following the manufacturer’s instructions. Fragmented mRNA was incubated with m6A antibody (Synaptic System, 202003) for immunoprecipitation. Then, immunoprecipitated mRNAs or Input was used for library construction with NEBNext ultra RNA library prepare kit for Illumina (New England Biolabs).The library preparations were sequenced on an Illumina Novaseq 6000 platform with a paired-end read length of 150 bp according to the standard protocols. The m6A peaks were detected by exomePeak R package (version 2.16.0). The motif search was detected by HOMER(version 4.9.1). Conclusion: The iBAT mRNA m6A profiles in Wtapflox/flox and BKO mice were characterized. To map the mRNA m6A modification caused by WTAP in iBAT, MeRIP-seq was performed in iBAT obtained from Wtapflox/flox and BKO mice.