Read mapping and counting

The mouse genome assembly (GRCm38.p6) was indexed using the STAR alignment tool (version 2.7). STAR –runMode genomeGenerate –genomeFastaFiles GRCm38.p6.genome.fa –sjdbGTFfile gencode.vM25.annotation.gtf –sjdbOverhang 79. Reads were then mapped to the mouse genome using the same tool. STAR –genomeDir –readFilesIn raw_reads.fastq –outFilterMultimapNmax 1 –outSAMtype BAM SortedByCoordinate. The quantification of the RNA-seq data was performed using featureCounts (version 2.0). featureCounts -s 0 -t -g -a gencode.vM25.annotation.gtf -M -O –fraction –maxMOp 1000 -o output.txt input.bam. The -t option in featureCounts was specified based on the exon and intron level quantification. In order to quantify reads based on introns, a custom annotation file was generated from the original mouse annotation file using the makeEISAgtfs script from the EISAcompR package (61) available at https://github.com/emarmolsanchez/EISAcompR.