title: GSM9010251: Pooled CD8 T cells, post yellow fever virus vaccination, Experiment 1, Sample 2, scGEX; Homo sapiens; RNA-Seq
library_construction_protocol: PBMCs were thawed from cryopreserved stocks and rested overnight at 1x10^6 cells/mL in cRPMI medium (RPMI 1640 Medium + 10% heat-inactivated FCS, 0.05 mM β-mercaptoethanol, 0.05 mg/mL gentamicin, 1.1915 g/L HEPES, 0.2 g/L L-glutamine, 100 U/mL Penicillin-Streptomycin). For experiment 1+2: 5-10^6 PBMCs per donor and timepoint were recovered and first stained with a cocktail of differentially conjugated anti-CD45 surface antibodies and individual hashtag antibodies (2.5 µL per 5x10^6 PBMCs of TotalSeq-C anti-human hashtag antibodies 1–8, Biolegend, 394661, 394663, 394665, 394667, 394669, 394671, 394673, 394675) for 30 min on ice. Afterwards, up to 8 individual samples were pooled and were later distinguished by individual CD45 color-barcoding at the sorter and by individual TotalSeq-C anti-human hashtag antibodies in the scRNAseq dataset. During the incubation, a dextramer cocktail containing 13 different dextramers (experiment 1) or only the A2/NS4B214-dextramer (experiment 2). For this cocktail, 1 µL per 5x10^6 pooled cells of dCODE dextramers® (immudex) and 100 µM d-biotin (1/500 of total dextramer volume) were pre-mixed in FACS-buffer (PBS + 0.5% BSA) to block free binding sites. The pooled samples (40-60x10^6 cells) were stained with 400-600 µL of the dextramer cocktail for 30 min on ice. Then, 400-600 µL of a cocktail containing vaibility dye ZombieNir, fluorescently-labelled antibodies for cell sorting (anti-CD8, -CD4, -CD19, -CD56) and DNA-barcoded CITE-seq antibodies (BioLegend: anti-CD45RA (304163), -CD62L (304851), -CD95 (305651), -CCR7 (353251), -CXCR3 (353251))was added and cells were stained for additional 30 min on ice. Single, live, CD19-, CD56-, CD4-, CD8+, dextramer+ lymphocytes were sorted on a FACS Aria II cell sorter (BD) in previously FCS-coated 1.5 mL tubes filled with FACS-buffer. For experiment 3: 10x10^6 PBMCs per donor and timepoint were recovered and stained with pre-preapred dextramer cocktail (2µL A2/NS4B214-dextramer, 0.4µL 100µM d-Biotin in 100µL FACS-buffer) for 30 min on ice. dextramer+ cells were then enriched by magnetic cell separartion using the MACS Mini Separator (Miltenyi Biotec) with anti-PE microbeads following manufacturer's instructions. Enriched cells were then incubated with a mix of ZombieNir viability dye, fluorescently labeled surface antibodies (Anti-CD18, -CD56, -CD4, -CD8 and differentially-labelled anti-CD45 for donor-separation during sorting), donor -specific TotalSeq-C anti-human hashtag antibodies (as indicated for experiment 1+2) and CITEseq antibodies (as indicated above) for 30 min on ice. Up to 8 samples were then pooled into one sample for sorting and single, live, CD19-, CD56-, CD4-, CD8+, dextramer+ lymphocytes were sorted on a FACS Aria II cell sorter (BD) in previously FCS-coated 1.5 mL tubes filled with FACS-buffer. For all experiments: Immediately after sorting, cells were loaded to a Chromium Next GEM Chip K (10X Genomics) and Chromium Next GEM Single-Cell 5′ kits v.2 were used to generate GEX, VDJ and CITEseq libraries according to the manufacturer's instructions (10X Genomics, 1000263, 1000256, 1000252, 1000286, 1000250, 1000215, 1000190). Libraries were sent to Novogene and sequenced on an Illumina NovaSeq platform with PE150 strategy. Library construction for scRNAseq was performed according to manufacturer's instructions (10x Genomics) for 5' GEX, VDJ and CITEseq, V2 chemistry dual index.