Transcriptome sequencing of rheumatoid arthritis patient-derived synovial fibroblasts with or without NRP2 knockdown

Synovial fibroblasts were isolated from synovial tissues of rheumatoid arthritis patients, passaged, and purified, and identified by flow cytometry. Cells from passages 3 to 8 were used for experiments. After transfection of siCon or siNRP2 via electroporation for 24 hours, the cells were stimulated with TNF for 2 or 20 hours, collected, and subjected to transcriptome sequencing. RNA-seq and data analysis were performed by Seqhealth Technology Co., Ltd. (Wuhan, China). Total RNA was extracted using TRIzol, and 2 μg was used to prepare stranded mRNA libraries with the KC-Digital™ Kit (Illumina-compatible) per manufacturer protocols. Raw data were quality-filtered (Trimmomatic v0.36) and adaptor-trimmed, followed by in-house deduplication to remove PCR/sequencing biases. Exon-mapped reads were quantified (featureCounts, Subread-1.5.1), RPKM-normalized, and analyzed for differential expression (edgeR v3.12.1; p<0.05, |FC|≥2).