ATAC-seq library for PBS-treated AM7754
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. Chondrocyte media was aspirated and cells were washed with PBS. To avoid changes associated with trypsinization, cells were directly lysed in the well as previously described(doi: 10.1016/j.cell.2020.12.025). Briefly, cells were washed twice with cold PBS followed by one wash with cold ATAC-seq resuspension buffer (RSB). Cells were lysed in RSB containing 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin for 10 min at 4C. After lysis the remainder of the Omni-ATAC protocol was performed(doi:10.1038/nmeth.4396). Following washes and  transposition with Tagment DNA TDE1 Enzyme (Illumina #20034197) reactions were cleaned up with DNA clean and concentrator kit (Zymo Research #D4014). Samples were preamplified using High-Fidelity 2X PCR Master Mix (New England Biolabs, #M0541L) and adapters (Illumina Nextera XT Index kit #FC-131-1001). The number of additional cycles was determined by quantitative PCR. Following a double-sided AMPure XP bead cleanup (Beckman Coulter #A63881), libraries were quantified using Qubit. Library quality and fragment distribution was visualized by Agilent TapeStation 4150. Prior to pooling, libraries were quantified with the KAPA library quantification kit (Roche #07960298001). Libraries were sequenced on Illumina NextSeq 500 sequencer (75-bp paired-end reads, high output kit Illumina #20022907) at the CRISPR core, University of North Carolina.