title: GSM6044094: RA patient 3; Homo sapiens; RNA-Seq
library_construction_protocol: RA and PsA patients (defined by the ACR and CASPAR Criteria respectively) were recruited from the Rheumatology Department, St. Vincent's University Hospital, UCD and Tallaght University Hospital, TCD.  RA and PsA patient synovial tissue samples from knee joints with active inflammation were obtained under local anaesthetic using Wolf 2.7 mm needle arthroscopy or ultrasound guided biopsy as previously described, please see table S1 for patient clinical characteristics 63.  RA and PsA patients had comparable moderate to high disease activity (DAS28 4.6 ± 1.1 and DAPSA 24.2 ± 4.9 respectively) and biopsies had lymphocyte infiltrates and lining layer hyperplasia as scored by a clinical pathologist. All patients gave fully informed written consent approved by the institutional Ethics Committee and research was performed in accordance with the Declaration of Helsinki.Synovial tissue single cell suspensions were generated following enzymatic and mechanical digestion of the synovial biopsies as described previously 27. Briefly, approximately 15 synovial biopsies per patient were digested using the GentleMACS Tumor Dissociation Kit, human (Miltenyi Biotech) as per manufacturers' instructions. Immediately after isolation, biopsies are washed with RPMI (Merck) before being placed in 4.7 ml RPMI supplemented with 200 μl of enzyme H, 100 μl enzyme R and 25 µl enzyme A in a gentleMACS™ C Tube followed by initial mechanical disruption of the tissue using program h_tumor_01 on a gentleMACS™ Dissociator. Samples are enzymatically digested for a total of 1 h at 37 °C under continuous rotation using the MACSmix Tube Rotator with further applications of the gentleMACS™ Dissociator at the halfway point and at the end of the 1 h incubation. The cell suspension is then passed through a 70 µM cell strainer. Viability of the cells is assessed with trypan blue exclusion staining and immediately cryopreserved in sterile filtered 10% DMSO/FBS at a concentration of 1x10ˆ6 cells per ml. Using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10X Genomics), cells were loaded onto the GEM Chips.  The 10X Genomics Chromium Next GEM Single Cell 3' user manual was followed for all steps to generate cDNA libraries for each sample. cDNA quantifications and quality control were determined using the Agilent TapeStation. Final libraries were normalized, quantified (Illumina/ROX low, Kappa Biosystems), pooled based on 40k reads/sample.  Pooled libraries were sequenced on the Illumina NovaSeq using and S2 NovaSeq 6000 Reagents v1 kit and a 100-cycle sequencing run.