title: Transcriptomic profiling of peripheral blood mononuclear cells from rheumatoid arthritis patients before tofacitinib treatment
design: Transcriptomic profiling of peripheral blood mononuclear cells from rheumatoid arthritis patients before tofacitinib treatment
library_construction_protocol: Fourteen female patients with active rheumatoid arthritis (RA) were enrolled. Patients received tofacitinib (Pfizer Inc., 5 mg per tablet) orally, 5 mg twice daily, for 8 weeks. Peripheral blood (20 mL) was collected from each patient in fasting state into EDTA anticoagulant tubes at two time points: before treatment (week 0) and after 8 weeks of tofacitinib therapy (week 8). Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation according to standard protocols. Isolated PBMCs were immediately resuspended in TRIzol reagent and stored at −80°C until RNA extraction. Patients were later stratified into responders and non-responders based on EULAR DAS28-defined clinical response after 8 weeks of treatment. PBMCs were collected from RA patients and stored in TRIzol at −80°C. Total RNA was extracted using TRIzol Reagent following the manufacturer's instructions. RNA concentration, purity, and integrity were assessed using a NanoDrop spectrophotometer. Two micrograms of total RNA per sample were used to construct RNA-seq libraries. Ribosomal RNA was depleted using the Epicentre Ribo-Zero™ rRNA Removal Kit (Human/Mouse/Rat). RNA was fragmented using divalent cations under elevated temperature. First-strand cDNA was synthesized using random primers, followed by RNA degradation with RNaseH. Second-strand cDNA was synthesized using DNA polymerase I with dUTP replacing dTTP. Double-stranded cDNA was purified, end-repaired, adenylated at the 3' ends, and ligated with sequencing adapters. USER enzyme was applied to degrade the second strand containing uracil. Size selection of 400–500 bp fragments was performed using AMPure XP beads. Libraries were PCR-amplified for 15 cycles and purified with AMPure XP beads. Library quality and fragment size distribution were assessed using the Agilent High Sensitivity DNA Kit on a Bioanalyzer 2100 system.