RNA-seq library for FN-f-treated 23-0062
Primary synovial fibroblasts were isolated from human synovial tissue of osteoarthritis patients who underwent total knee arthroplasty at the University of North Carolina Hospital, Hillsboro, North Carolina, USA. For synovial fibroblast isolation, tissue was minced, enzymatically digested, passaged at confluency, and used for experiments at passage 3, as previously described (PMID: 39485774). Cells were maintained in culture in a complete medium consisting of DMEM (Gibco, 11965-092) supplemented with 10% fetal bovine serum (FBS) (Avantor, 97068-085), penicillin-streptomycin (100 U/ml) (Gibco, 15140-122), and 1% GlutaMAX (Gibco, 35050-061) at 37ºC and 5% CO2. For fibronectin fragment (FN-f) stimulation experiments, serum starvation was required for 2 hours, and synovial fibroblasts were washed twice with PBS. Synovial fibroblasts were then treated with 1 µM purified recombinant human FN-f (FN7-10), prepared as previously described and stored as aliquots at -80 degrees C in PBS (PMID: 37906629), or treated with PBS alone as a control for 18 hours. The media was then removed, and the cells were washed once with PBS before lysis using RNeasy Lysis Buffer (Qiagen). Lysates were stored at -80 °C. RNA was extracted using the RNeasy kit (Qiagen #74104) according to the manufacturer's recommendations. On-column DNase digestion was performed during the extraction. Samples were quantified with the Qubit RNA High Sensitivity Assay Kit (Thermo Fisher Scientific #Q32582), and RNA integrity number (RIN) was obtained using the Agilent TapeStation 4150. RNA-seq libraries were prepared using the KAPA RNA HyperPrep Kit with RiboErase (HMR).
NIAMS-phs003999
FACT_23-0062_FNF_synovium_1