The effects of WTAP on m6A mRNA modification in the heart [meRIP-seq]

Through m6A mRNA-profiling, we aim to characterize the m6A mRNA changes in the hearts of Wtapflox/flox and Wtap-CKO mice. To map the mRNA m6A modification caused by WTAP in the hearts, MeRIP-seq was performed in the hearts of Wtapflox/flox and Wtap-CKO mice. Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from the hearts of Wtapflox/flox and Wtap-CKO mice at 4 days old. A total of 300 μg RNAs were pooled from seven Wtapflox/flox mice and eight Wtap-CKO mice, respectively. Fragmented RNA (~100 nt) was incubated for 2 hr at 4℃ with anti-m6A polyclonal antibody (Merk Millipore) in the immunoprecipitation experiment. Then, immunoprecipitated RNAs or Input was used for library construction with Ovation SoLo RNA-Seq System Core Kit (NuGEN). The library preparations were sequenced on an Illumina Novaseq platform with a paired-end read length of 150 bp according to the standard protocols. After mapping reads to the reference genome, exomePeak R package (version 2.16.0) was used for the m6A peak identification in each anti-m6A immunoprecipitation group with the corresponding input samples serving as a control, and q-value threshold of enrichment of 0.05 was used for all data sets. The m6A-enriched motifs of each group were identified by HOMER (version 4.9.1). The heart mRNA m6A profiles in Wtapflox/flox and Wtap-CKO mice were characterized.