Informatics

The standardised RNA sequencing analysis pipeline, nf-core/rnaseq v3.3, was used for quality control, alignment and quantification. Details can be found at https://nf-co.re/rnaseq/3.3. In short, quality control was carried out with FASTQC, adapter and quality trimming with TrimGalore, alignment with STAR and quantification with Salmon. Default settings were used except for STAR “seedSeachStartLmax 25” to increase sensitivity of mapping for 40 bp reads, and TrimGalore “–trim_nextseq 20” to set the Phred score threshold at 20. Reads were aligned to the human genome GRCh37. Count files were imported into R for differential expression analysis with DESeq2 using the default Wald test. P values were adjusted using the Benjamini-Hochberg (BH) method and the threshold for differential expression was BH-adjusted P < 0.05. Gene ontology enrichment and protein–protein interactions analyses were performed using (1) Qlucore Omics Explorer 3.8 (https://qlucore.com/), and ShinyGo 0.77 [26]. Multi- and two-group comparison of RNA sequence variables were performed at False Discovery Rates (FDR, Q) < 0.05 as reported in figures. Analysis were performed in April 2023. ShinyGO algorithm search parameters of interrogation comprised: Species: Human. FDR cutoff: 0.05; # pathways to show: 20; Pathway size minimum: 2; Pathway size maximum: 2000. Other Options: Redundancies removed, Pathways abbreviated. STRING Pathway Parameters: Human. Display to include up to all genes interrogated.