Mouse Transcriptome Sequencing Workflow

Using a molecular barcode, the data was automatically demultiplexed using the Illumina bcl2fastq2 Conversion Software v2.20. FastQ files underwent two rounds of quality control, pre-trimming, and post-alignment, using Fast Q20 v0.11.7 (Andrews, 2010 ). Removal of Illumina adapters and low-quality sequences was performed with Trimmomatic v0.38 (Bolger et al., 2014 (link)). Reads of length <15 bases, as well as leading and/or trailing bases with quality <3 or no base call, and bases with average quality <15 in a 4-base sliding window were removed. Alignment was performed with HISAT2 v2.1.0 (Kim et al., 2019 (link)) using the mouse genome assembly mm10 (Mus musculus, GRCm38). Mapped reads (primary alignments) were sorted by read name using SAMtools v1.8 (Li et al., 2009 (link)), and read counts were calculated with HTSeq v0.11.2 (Anders et al., 2015 (link)).