m6ARIP-Seq of liver in Wtapflox/flox and hepatocyte-specific Wtap knockout (HKO) mice

Purpose: Through mRNA m6A-profiling, we aim to characterize the mRNA m6A changes in the liver obtained from Wtapflox/flox and Wtap-HKO mice. Methods: Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from liver of Wtap flox/flox and Wtap-HKO mice at 8 weeks old. Each sample (300 μg total RNA) was pooled from 5 mice for each group. Two independent biological replicates for each group were used for m6ARIP-seq. 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 mRNA m6A profiles in the liver of Wtapflox/flox and Wtap-HKO mice were characterized. To map the changes of mRNA m6A modification caused by hepatic deletion of Wtap, MeRIP-seq was performed in liver obtained from Wtapflox/flox and Wtap-HKO mice.