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.