RNA-seq Data Analysis Pipeline

Sequencing libraries were prepared using a TruSeq Stranded mRNA Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions and were sequenced by the Illumina NovaSeq 6000 system (performed by Genomics BioSci. & Tech., Taipei, Taiwan). Read quality was evaluated by FastQC and adaptor sequences were trimmed using cutadapt. Qualified reads were aligned to the human reference genome GRCh38 using STAR (v. 2.7.2) [15 (link)] and read counts for individual genes annotated based on GENCODE (v. 28) were subsequently determined using featureCounts [16 (link)]. Differential expression analysis was performed using limma [17 (link)] with TMM (trimmed mean of M-values) normalization. For pre-ranked GSEA, a ranking metric was calculated for each gene as R = sign(log₂FC)* – log₁₀(p-value), where both log₂FC and p-value were determined by limma. Pre-ranked GSEA was performed using a curated collection of gene sets from MSigDB. The significant gene sets were constructed as an enrichment map [18 (link)] using an in-house script and visualized using Cytoscape.