Library preparation and RNA-sequencing

RNA seq libraries were obtained starting from 500 ng of total RNA following Illumina TruSeq Stranded TotalRNA preparation protocol. Sequencing was performed using Illumina NEXSeq high-output cartridge (double-stranded, reads length 75bp-2 ×75).

A sequencing depth of at least 60 million reads for each sample was guaranteed.

Sequencing quality was assessed using the FastQC v0.11.8 software (www.bioinformatics.babraham.ac.uk/projects/fastqc/), showing on average a Phred score per base >34 in each sample. Raw sequences were then aligned to the human reference transcriptome (GRCh38, Gencode release 30) using STAR version 2.7²³ and gene abundances were estimated with RSEM algorithm (v1.3.1)²⁴. Differential expression analysis was performed using DESeq2 R package²⁵, considering a False Discovery Rate (FDR) of 10% and excluding genes with low read counts. Heatmap representation and unsupervised hierarchical clustering with a complete linkage method were exploited to graphically depict differentially expressed genes (FRD < 0.1).

Significant genes underwent enrichment analysis, performed on Gene Ontology biological processes, KEGG and Reactome pathways databases via enrichR package²⁶, using a significance threshold of 0.05 on p-value adjusted by Benjamini–Hochberg correction for multiple testing.