The effects of ALKBH5 on m6A mRNA modification in the liver [MeRIP-seq]

Through m6A mRNA-profiling, we aim to characterize the m6A mRNA changes in the liver between Alkbh5flox/flox and Alkbh5-HKO mice. To map the mRNA m6A modification caused by ALKBH5 in the liver, MeRIP-seq was performed in the livers of Alkbh5flox/flox and Alkbh5-HKO mice. Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from livers of Alkbh5flox/flox and Alkbh5-HKO mice at 9 weeks old. A total of 300 μg RNAs were pooled from three mice.Fragmented mRNA (~100 nt) was incubated for 2 hr at 4oC with anti-m6A polyclonal antibody (Synaptic Systems) in the immunoprecipitation experiment. 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 Novaseq6000 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 liver mRNA m6A profiles in Alkbh5flox/flox and Alkbh5-HKO mice were characterized.