Cell ranger v2.0.2

ScRNAseq sequencing was performed on ultrasound-guided joint biopsies of hand (n = 8) or knee (n = 6) joints of RA patients (Patient’s characteristics in Supplementary Table S1). Tissues were processed as previously described^{42 (link)}. In brief, tissues were mechanically minced and enzymatically digested using Liberase TL (Roche). 10’000 unsorted synovial cells (viability >80%) per patient were prepared for single cell analysis using the Chromium Single Cell 3’ GEM, Library and Gel Bead Kit v3.1, the Chromium Chip G Single Cell Kit and the Chromium controller (all 10× Genomic). Libraries were sequenced on the Illumina NovaSeq6000 instrument to a sequence depth of 20,000 to 70,000 reads per cell. CellRanger (v2.0.2) from 10× Genomics was used to demultiplex, align the reads to Ensembl reference build GRCh38.p13 and collapse unique molecular identifiers (UMIs). The standard Seurat protocol^{73 (link)} was used for further analysis. Gene expression between hand and knee was compared. Pathway enrichment analyses of genes differentially expressed between SF from hands and knees were performed using Enrichr (all genes with FDR < 0.05, log fold change +/−1)⁷⁴ . The scatter dot plots were created with the Enrichr Appyter.

Based on the previous studies,²⁰ (link) four typical synovial tissues (two AF group patients and two control group patients) were mechanically minced and enzymatically digested with Liberase TL (100 g/mL; Roche) and DNAse I (100 g/mL; Roche) for 30 min at 37°C. Red Blood Cell Lysis solution was used to lyse erythrocytes after fetal calf serum was used to stop the digestion process (Milteny Biotec). The LUNA automated cell counter was used to wash and count the cells (Logos Biosystems). Using the Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3, the Chromium Chip B Single Cell Kit (10 Genomics), and the Chromium controller (all 10 Genomics), a total of 15 000 unsorted synovial cells per patient were prepared for single cell analysis. On the Illumina NovaSeq instrument, libraries were sequenced to a depth of 20 000–70 000 reads per cell. The reads were demultiplexed, and aligned to the Ensembl reference build GRCh38.p13, and the unique molecular identifiers were collapsed using CellRanger (V.2.0.2) from 10X Genomics.