MeRIP-m6A-Seq, RNA-Seq and data analysis

The m⁶A-Seq was performed by Cloudseq Biotech Inc. (Shanghai, China) according to the published procedure (31) with slight modifications. Briefly, 5 μg of fragmented mRNAs were saved as input control for RNA-seq, 500 μg of fragmented mRNAs were incubated with 5 μg of anti-m⁶A polyclonal antibody (Synaptic Systems, 202003) in IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris–HCl, pH 7.4) for 2 h at 4°C. The mixture was then immunoprecipitated by incubation with protein-A beads (Thermo Fisher) at 4°C for an additional 2 h. Then, bound mRNAs were eluted from the beads with N⁶-methyladenosine (BERRY & ASSOCIATES, PR3732) in IPP buffer and then extracted with Trizol reagent (Thermo Fisher) by following the manufacturer's instruction. Purified mRNAs were used for RNA-seq library generation with NEBNext^® Ultra™ RNA Library Prep Kit (NEB). Both the input sample (without immunoprecipitation) and the m⁶A IP sample were subjected to 150 bp paired-end sequencing on Illumina HiSeq sequencer.

Paired-end reads were harvested from Illumina HiSeq 4000 sequencer, and were quality controlled by Q30. After 3′ adaptor-trimming and low quality reads removing by cutadapt software (v1.9.3). The reads were aligned to the reference genome (UCSC HG19) with Hisat2 software (v2.0.4). Methylated sites on RNAs (peaks) were identified MACS software. Differentially methylated sites on RNAs were identified by diffReps. These peaks identified were mapped to transcriptome using home-made scripts.