GSM7914563: DK2_high; Homo sapiens; RNA-Seq
Synovial tissue was digested in 3 mL of complete RPMI (10% FBS, 1% pen/strep) containing 400 μg/ml of LiberaseTM (Roche, Basel, Switzerland) and 400 μg/mL of DNAse I for 30 minutes at 37°C while rotating at 14 rpm. Next, 100 mM EDTA was added to stop enzymatic digestion and cells were washed twice with DMEM prior to trypan exclusion for total live cell counts and viability. Cells were then aliquoted into 1.0-2.0 million cells per 1 mL of CS10 (Stemcell Technologies, Vancouver, BC, Canada), frozen at -80°C overnight in a controlled rate cell freezing container (Mr. FrostyTM) and transferred to a liquid nitrogen vapour phase storage container until use. Synovial single cell suspensions from each patient were rapidly thawed from cryopreservation and underwent trypan exclusion to confirm viability >75% prior to library prep. Cells were prepared for cDNA amplification and chromium library construction using the 10x Chromium Next GEM Single Cell 5' Library and Gel Bead Kit v1.1 according to manufacturer's protocol (10x Genomics)