title: GSM5830320: Cytokine-stimulated 3.3_TCR; Homo sapiens; OTHER
library_construction_protocol: PBMCs were isolated from fresh whole blood by density gradient centrifugation (Lymphoprep, Axis-Shield) at 2000 rpm (931 g) for 30 min with no brake. Cells were cryopreserved (90% FCS, 10% DMSO [Sigma-Aldrich]) in liquid nitrogen. A Pierce streptavidin coated high capacity flat-bottom 96-well plate (Thermo Fisher Scientific) was coated with 50 μl biotinylated MR1/5-OP-RU monomer (NIH Tetramer Core Facility) at 10 μg/ml in PBS overnight at 4°C. Cryopreserved PBMCs from three healthy female donors were thawed in complete medium. CD8+ T cells were enriched using CD8 MicroBeads (Miltenyi Biotec) following manufacturer's instructions. Enriched CD8+ T cells were washed in complete medium and resuspended at 10 million cells/ml. One million cells per well were added to the appropriate 96-well plate (MR1/5-OP-RU-coated plate for the TCR stimulation condition, round-bottom plate for the unstimulated and cytokine stimulation conditions). IL-12 (50 ng/ml; R&D Systems) and IL-18 (50 ng/ml; R&D Systems) were added to the cytokine stimulation condition, αCD28 (1 μg/ml; clone: CD28.2; BioLegend) to the TCR stimulation condition, and complete medium to the unstimulated condition (final volume 200 μl in all wells). Cells were incubated for 20 hours at 37°C, 5% CO2. Surface staining was performed in Brilliant Stain Buffer (BD Biosciences) for 30 min at 4°C. Cells were washed twice in PBS with 0.5% BSA, resuspended in pre-sort buffer containing 3-5 nM SYTOX Green Nucleic Acid Stain (Thermo Fisher Scientific), and incubated for 20 min at 4°C. MAIT cells (CD3+CD8+CD26+CD161hiVα7.2+) were sorted on a BD FACSAria III with a 70 μm nozzle. Sorted cells were collected in RPMI-1640, 10% FBS, 25 mM HEPES. Sort purity was > 99%. scRNA-seq and scTCR-seq libraries were generated using Chromium Single Cell V(D)J Reagent Kits (v1.0 Chemistry) following manufacturer's instructions.