title: GSM6797155: OA Cartilage single cell RNAseq, Donor2; Homo sapiens; RNA-Seq
library_construction_protocol: All human tissues were obtained with approval by the Scripps Human Subjects Committee. Normal human knee joints were obtained from Lifesharing, San Diego from donors without history of joint injury or disease. OA tissues were obtained from donors undergoing total knee arthroplasty. Cartilage samples were minced with a #21 Feather disposable scalpel, and then digested in Dulbecco's Modified Eagle Media (DMEM) supplemented with 1% Anti-Anti and 2% collagenase type II with 100 rpm shaking at 37°C for 2 hours.  Cells were passed through a 100 µm filter followed by passage through a 40 µm filter. Filtered cells were spun down at 1200 rpm for 5 minutes at room temperature. The supernatant was removed, and the remaining cell pellet was resuspended in PBS supplemented with 5% CS and 5 mM EDTA. Resuspended single cells were then treated with DNase I for 15 minutes at room temperature. Single cells were then spun down at 1200 rpm for 5 minutes at room temperature. The supernatant was removed, and the remaining cell pellet was resuspended in PBS supplemented with 0.04% BSA. Whole meniscus was minced with a #21 Feather disposable scalpel, and then digested in Dulbecco's Modified Eagle Media (DMEM) supplemented with 1% Anti-Anti and 1% collagenase type II with 100 rpm shaking at 37°C for 30 minutes.  Cells were passed through a 100 µm filter followed by passage through a 40 µm filter. Filtered cells were spun down at 1200 rpm for 5 minutes at room temperature. Cells were then resuspended in DMEM supplemented in 10% CS, 1% Anti-Anti and 1% PSG and stored at 37°C. The digestion, spin down and filtration steps were repeated for 1 hour and then 2 hours, for a total of 3.5 hours of digestion. The stored cells were combined and then spun down at 1200 rpm for 5 minutes at room temperature. The supernatant was removed, and the remaining cell pellet was resuspended in PBS supplemented with 5% CS and 5 mM EDTA. Single cells were then spun down at 1200 rpm for 5 minutes at room temperature. The supernatant was removed, and the remaining cell pellet was resuspended in PBS supplemented with 0.04% BSA. For both tissues, the Invitrogen Countess II FL automated cell counter was used to quantify single cells and determine cell viability. Live cells were determined by trypan blue staining. If >70% cell viability was confirmed, the single cell suspension was diluted to a concentration of 1x106 cells/mL and cells were then loaded onto a Chromium Next GEM chip G (10X Genomics). Single cell RNAseq was performed using the 10X Genomics Chromium technology. Briefly, cells were partitioned into droplets using the Chromium Controller with the Next GEM Single Cell 3' Reagent Kit v3.1 using dual indexing. Next, cells were lysed and the polyadenylated transcripts reverse transcribed to generate barcoded cDNA following the recommended 10X Genomics protocol. The quality of both the cDNA and the final libraries were assessed using the Agilent Bioanalyzer 2100 following the guidance provided by the 10X Genomics user guide.