RNA sequencing

Quality assessment of the raw fastq reads of the sample was performed using FastQC v.0.11.9 (default parameters) and summarised using MultiQC v.1.9. The raw fastq reads were preprocessed using Fastp v.0.20.1(parameters: --qualified_quality_phred 30 --trim_front1 9 --trim_front2 9 --trim_tail1 0 --trim_tail2 0 --length_required 50 –correction --trim_poly_g), followed by quality re-assessment using FastQC and summarised using MultiQC. The splicesites and exons were extracted from mouse genome (NCBI: mouse genome (GRCm39), and indexed using HISAT2 v.2.2.1 (Parameters: hisat2-build –ss genome_splicesites.txt --exon genome_exons.txt). The indexed genome was then mapped against processed reads using HISAT2 (parameters: --dta --mm --summary-file --un-conc-gz --al-conc-gz). Aligned reads were converted to bam and sorted using Samtools v.1.7 (sort parameters: sort -l 9). The Aligned reads from individual samples were quantified using feature count v. 0.46. 1 (parameters: -g gene_id -F GTF -p) to obtain gene counts. These gene counts were used as inputs to DESeq2 [18] for differential expression estimation (parameters: threshold of statistical significance --alpha 0.05; p-value adjustment method: BH).

Gene Ontology (GO) analysis was done using gProfiler (parameter: organism= Mus musculus) online server. Pathway enrichment analysis was done using InnateDB web server. Gene ontology and metabolic pathway enrichment analysis were also performed using Network Analyst (https://www.networkanalyst.ca/) online server [19]. The featureCout generated count file was converted to NetworkAnalyst input format and processed using default parameters. Geneset Network Analysis was done using Moderated Welch’s T test.