RNA-seq library for FN-f-treated AM7280
Chondrocytes from human talar cartilage of a deceased tissue donor with no known history of arthritis were isolated via enzymatic digestion and cultured in monolayer using protocols as previously described (doi: 10.1016/j.joca.2020.09.006)  Chondrocytes were given 4 days of recovery from digestion and maintained in cell culture medium consisting of DMEM/F12 supplemented with 10% FBS (VWR Seradigm; #97068-085) before experiments. For FN-f treatment, serum-containing media was first removed, and the cells washed twice with PBS before being serum-starved in DMEM/F12 for 2 hrs. Chondrocytes 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 (doi: 10.1126/scisignal.adf8299), or treated with PBS alone as a control for 18 hours. The media was then removed, and the cells washed once with PBS before lysis using RNeasy Lysis Buffer (Qiagen). Lysates were stored at -80° until library preparation. RNA was extracted using the RNeasy kit (Qiagen #74104) according to the manufacturer's recommendation. 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 was submitted to the New York Genome Center for RNA-seq library preparation and sequencing.