RNA-Seq data analysis
The initial raw sequencing data underwent preprocessing steps. Trimmomatic (version 0.36) was employed to filter out low-quality reads and remove reads contaminated with adapter sequences. Clean reads were further processed using in-house scripts to address duplication bias introduced during library preparation and sequencing.
The de-duplicated sequences were aligned to the reference genome of Homo sapiens using STAR software (version 2.5.3a) with default parameters. Reads mapped to the exon regions of each gene were counted using featureCounts (Subread-1.5.1; Bioconductor), and Reads Per Kilobase per Million mapped reads values were calculated. Differential gene expression analysis between groups was conducted using the edgeR package (version 3.12.1). Statistical significance of gene expression differences was assessed using a p-value cutoff of 0.05 and a fold-change cutoff of 2. GO analysis and KEGG enrichment analysis for differentially expressed genes were performed using KOBAS software (version: 2.1.1). A p-value cutoff of 0.05 was used to determine statistically significant enrichment.