Hi-C library for FN-f-treated AM7683 replicate 1
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. 8 million chondrocytes were cultured in DMEM/F-12 media, serum-starved for 2 hours, and treated with PBS or FN-f. After 18 hours of treatment, the media was removed from the plate. Cells in each plate were crosslinked in 10% formaldehyde in DMEM/F-12 media and incubated for 10 minutes on a rocker. To quench, 2M Glycine was added as a final concentration of 0.2M and incubated for 5 minutes on the rocker. The supernatant was removed, the cells were resuspended with 10mL cold PBS, collected into a 15mL tube, and spun down at 2500 rpm, 4°C for 5 minutes. The pellets were resuspended with 1mL PBS, transferred to 1.5mL microcentrifuge tube, and spun down at 900g, 4°C for 5 minutes. The pellets (~8 million cells) were flash frozen in liquid nitrogen and stored at -70 °C. The cells were thawed and in situ Hi-C was performed as described in Rao et al. (2014) (doi: 10.1016/j.cell.2014.11.021).