GSM6605183: OA_Knee_5; Homo sapiens; ATAC-seq
Fresh cartilage tissue from proximal (hip, n=6) and distal (knee, n=6) human fetal femurs at 12 post conception weeks, was dissected and provided by the HDBR. Adult articular cartilage was dissected from hip (n=5) and knee (n=5) joint arthroplasty obtained as described above. For chondrocyte isolation, the cartilage was digested in 1% collagenase solution (Sigma Aldrich) for 3h (fetal) or 16h (arthroplasty) at 37˚C. Cells were washed with 1xPBS, re-suspended in 5%FBS-DMEM and counted. Nuclei were isolated from 50,000 cells by 3min incubation on ice in lysis buffer (5M NaCl, 1M MgCl2, 1M Tris-HCl (pH 7.5), 10% w/v NP-40 (Roche), 10% w/v Tween 20 (Roche) and 1% w/v Digitonin (Promega). Isolated nuclei were re-suspended in a transposase mix (25μl 2xTD Buffer, 2.5μl TDE1 enzyme (Nextera Tn5 transposase, Illumina), 0.5μl 1% w/v Digitonin, 0.5μl 10% w/v Tween 20, 16.5μl 1xPBS and 5μl nuclease-free H2O) and incubated for 30min at 37˚C, 1000rpm. DNA was purified using MinElute PCR Purification kit (Qiagen) according to manufacturer's instructions and eluted in 10μl H2O. The purified DNA was partially amplified by PCR in a 50μl reaction using the NEB Next High-Fidelity PCR Master Mix (New England Biolabs, Hitchin, UK) and primer Ad1_noMX in combination with barcoded primers (Supplementary Material, Table S15). A 5μl aliquot of the reaction was amplified by qPCR and the remaining 45μl was amplified by PCR for an additional number of cycles, calculated for each sample based on the qPCR readings, to avoid amplification to saturation. The libraries were purified using AMPure XP magnetic beads (Beckman Coulter).