title: 4040_ST
organism: Homo sapiens
description: Protocols: Synovial tissue was obtained from knee joints of PsA patients prior to commencement of any immunomodulatory therapy including systemic corticosteroids by a minimally invasive, ultrasound-guided technique with written informed consent. Protocols for these procedures were approved by relevant research ethics committees (reference number 12/NE/0251 Newcastle and West Midlands Black Country Research Ethics committee 07/H1203/57 Birmingham). Upon collection of biopsies, they were immediately cryopreserved in cryopreservation media Cryostor CS10 (Sigma - MERCK). Upon thawing, samples were digested in digestion buffer cocktail (50ug/ml Liberase TM, Sigma and 40ug/ml DNaseI, Roche, in RPMI) in a 37°C water bath for 30 minutes with continuous stirring with a magnetic stir bar in a U-bottom polystyrene tube (12 x 75 mm2). At 15 mins during the digestion, samples were passed gently 10 times through a 14G syringe needle for additional mechanical disruption. After digestion, the cells were filtered using a 70um filter and washed prior to staining with antibodies. Following digestion, cells were counted by trypan blue to assess cell quantity and viability. Cell suspensions were stained with antibodies against CD45-APC (2D1), CD31-PeCy7 (WM59), CD326- PeCy7 (9C4), CD235a- PeCy7 (HI264), CD117- PeCy7 (104D2). All antibodies were from Biolegend except the CD45 antibody which was from BD Biosciences. DAPI (Sigma) was added to the stained cells immediately prior to FACS sorting to isolate live leukocytes prior to loading onto separate channels of the 10x Chromium Controller. Standard 10x protocols for the kit were used to load cells into droplets containing barcodes. Cells were captured and cDNA preparation performed according to the Single Cell 5′ Protocol recommended by 10x Genomics.