title: Single cell TCR sequencing (scTCRseq) and whole transcriptome analyses (scRNAseq) of CD8+ T cells from synovial fluid (SF) of HLA-B*27:05 positive patients with SpA (SpA-B27pos) and controls
design: Cells were stained and sorted using the following procedure: 10min at 4C with Fc-block (Miltenyi #130-059-901) at 1/50 dilution in FACS buffer (PBS with 2% FBS), followed by the surface antibody staining mix at a total of 100ul FACS buffer for 30min at 4C: anti-human CD45RO-FITC (#11-0457-42) at 1/40 dilution, anti-human CCR7-APC (#17-1979-42) at 1/40 dilution, anti-human CD3-AF700 (#56-0037-42) at 1/50 dilution, Fixable Viability Dye eFluor 780 (#65-0865-14) at 1/1000, anti-human CD4-Pacific Blue (MHCD0428) at 1/25, all purchased from ThermoFisher, as well as anti-human CD8-PerCP (344708) at 1/50 from BioLegend. Antigen-experienced CD8+ T cells consisting of effector memory (CD45RO+, CCR7-), effector (CD45RO-, CCR7-) and central memory (CD45RO+, CCR7+) cells were collected using a FACS Aria Fusion flow cytometer (BD Biosciences). The Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 with User Guide Rev F was used. 20.6l of a 1200 cells/l suspension were used as input for a target recovery of 10,000 cells. The protocol was followed with minor alterations: TE buffer (Affymetrix #J75793) was used instead of elution buffer EB