RNA-seq and differential expression genes (DEG) analysis

PC3 cells were transfected with either siRNA targeting NOL10 or a negative control siRNA, incubated for 48 hours under standard cell culture conditions, with two biological replicates. Total RNA was extracted using Trizol reagent (#15596018, Thermo Fisher Scientific). RNA-seq libraries were prepared using the Stranded mRNA-seq Lib Prep Module (RK20349, Abclonal). The quality of libraries was assessed using LabChip Touch, and sequencing was conducted at Annoroad Company with Illumina sequencing platforms.

Raw sequence data were preprocessed using FastQC (v.0.11.9) (www.bioinformatics.babraham.ac.uk/projects/fastqc/) for quality assessment. AdapterRemoval (v.2.3.2)⁶⁰ was used for quality trimming and adapter removal with default parameters. The processed reads were aligned to the human genome (hg38) using STAR (v.2.7.9a)⁶¹ and the aligned BAM files were sorted using SAMtools (v.1.13)⁶². HTSeq (v.0.13.5)⁶³ was employed to quantify aligned sequencing reads against UCSC gene annotation with the parameters “-s reverse, -i gene_id”. DESeq2 (v.1.30.1)⁶⁴ was used for DEG analysis from the read count matrix. Genes with low expressions (<5 cumulative read count across samples) were filtered out. An adjusted P value < 0.05 was applied to generate the list of differentially expressed genes. DEGs were ranked according to their fold change. Statistical tests were applied to control or treatment to ensure high correlations between technical replicates. Data normalization was performed using the variance Stabilizing Transformation (VST) method. A heatmap presenting DEGs between siRNA control and siRNA NOL10 samples was generated using the R package “pheatmap” (v.1.0.12). Detailed information about the software and algorithms used is provided in Supplementary Table 5.