RNA isolations were performed using the RNeasy mini kit (Qiagen) according to the manufacturer’s procedure, including homogenization using the QIAshredder kit (Qiagen). DNase treatment was performed on-column to eliminate DNA contamination (Qiagen). Concentration and quality of the total extracted RNA was checked via the Quant-it Ribogreen RNA assay (Life Technologies) and the RNA 6000 nano chip (Agilent Technologies). The QuantSeq 3′ mRNA library prep FWD kit (Lexogen) was used for library preparation. Library QC was performed using the high sensitivity DNA chip (Agilent technologies). Sequencing was performed on the NextSeq 500 SR 76 high output system (Illumina). All sequencing data was deposited in GEO under the following accession number: GSE201012.
To determine the difference in gene expression of m6A-methylated transcripts compared to unmethylated transcripts, differential expression was determined for transcripts binned by the amount of m6A residues they contain. Reads were aligned to the Sscrofa11.1 genome using STAR (Dobin et al., 2013 (link)). Differential expression was determined using DESeq2 (Love et al., 2014 (link)).
To determine the difference in gene expression of m6A-methylated transcripts compared to unmethylated transcripts, differential expression was determined for transcripts binned by the amount of m6A residues they contain. Reads were aligned to the Sscrofa11.1 genome using STAR (Dobin et al., 2013 (link)). Differential expression was determined using DESeq2 (Love et al., 2014 (link)).