GSM9393394: synovium_time0_Control_KA15; Equus caballus; RNA-Seq
Synovial biopsies (approximately 2 mm x 10 mm samples) were obtained arthroscopically using ferris-smith rongeurs at time of surgical model induction at baseline prior to osteochondral fragment induction and at end-term week 10 immediately postmortem. Tissue samples were placed in complete media with Dulbecco's Modified Eagle's Medium (DMEM; Corning, catalogue #10-014-CM) with Fetal Bovine Serum (FBS; PEAK SERUM, Wellington, CO, USA). The tissue was washed with PBS and minced into 1mm pieces in a 6 well plate Type 2 collagenase (ThermoFisher, Waltham, MA, USA) in Hank's Balanced Salt Solution (HBSS; Corning, Durham, NC, USA) which was used at 0.4 U/mL. Tissue digestion was conducted in a CO2 incubator at 37 ºC for 60 minutes. Samples were agitated every 20 minutes with a pipette. Following digestion, the digested homogenate was passed through a strainer then transferred to a 15 mL conical and centrifuged at 526 x g for 5 min. Pelleted cells were washed twice with PBS then transferred to microcentrifuge tube. The microcentrifuge tube with the sample was spun at 100 x g for 15 minutes at 8 ºC to remove dead cells. The supernatant was discarded, and the cells were lysed in RLT buffer (Qiagen, catalogue # 79216) with 1% ß-Mercaptoethanol (BME; EMD Millipore Corp, Billerica, MA, USA) and stored at -80 ºC until RNA extraction. Library preparation for synovium was conducted with Tecan/Nugen Universal Plus mRNA-seq Poly A prep kit .