N6-methyladenosine Mapping by MeRIP-Seq

MeRIP-Seq was performed according to the published procedure (42 (link)). Briefly, fragmented mRNA was incubated with anti-m⁶A polyclonal antibody (Synaptic Systems, 202003) in immunoprecipitation buffer for 2 h at 4 °C and then immunoprecipitated by incubation with protein-A beads (Thermo Fisher) for another 2 h at 4 °C. Bound RNA was eluted from the beads with m⁶A (BERRY & ASSOCIATES) and extracted with the TRIzol reagent (Invitrogen). Fragmented mRNA without immunoprecipitation was used as input control. RNA-seq libraries were generated with the Next® Ultra™ II Directional RNA Library Prep Kit (New England Biolabs). Sequencing data were obtained from the HiSeq 2500 system (Illumina). After quality control, clean data were aligned to the reference genome (UCSC mm10) with Hisat2 software v2.2.1 (71 (link)). The read alignment on the genome was visualized by using the IGV tool v2.14.0 (74 (link)). MACS software v3.0.0a7 (75 (link)) was used for m⁶A peak calling with the significance cutoff q-value < 0.05. Peaks were annotated as located in 5′-UTR, CDS, 3′-UTR, intronic region and intergenic region. The metagene profile was drawn by the R package Guitar v2.12.0 (76 (link)). Motifs in m⁶A peaks were identified using HOMER v4.7 (77 (link)).