GSM9301199: pooledCells_scGEX2; Homo sapiens; RNA-Seq
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 each donor, 2x10^6 PBMCs were stimulated with stimulated with 11aa overlapping 15-mer PepMixTM SARS-CoV-2 spike glycoprotein peptide pool (JPT, PM-WCPV-S-2) at a concentration of 1 µg/mL for 4 h at 37°C. For the unstimulated condition, PBMCs were cultured in cRPMI medium and respective dilution of solvent DMSO. After stimulation, cells were washed with cold FACS buffer and stained with surface antibodies (anti-CD19-PE/CF594, anti-CD56-FITC, anti-CD8-APC, anti-CD4-PE, anti-CD62L-PacificBlue, anti-CD45RA-PerCP-Cy5.5). Additionally, individual hashtag antibodies (2.5 µL per 5x106 PBMCs of TotalSeq-C anti-human hashtag antibodies 1–8, BioLegend, 394661, 394663, 394665, 394667, 394669, 394671, 394673, 394675) were included for sample multiplexing. All samples were incubated for 30 minutes on ice, followed by two washes with cold FACS buffer. Live/dead discrimination was performed using PI (1:200) immediately before sorting. Single, live, CD19- and CD56-negative, CD4- or CD8-positive, non-naïve (defined as CD45RA-positive and CD62L-negative, CD45RA-negative and CD62L-negative, or CD45RA-negative and CD62L-positive) lymphocytes were sorted in previously FCS-coated 96-well U-bottom plates filled with FACS buffer. Eight different samples were pooled prior to sequencing and later distinguished using the unique TotalSeq-C hashtags during scRNAseq analysis. Cells were sorted on a FACS Aria II cell sorter (BD). 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 (Cambridge, UK) and sequenced on an Illumina NovaSeq platform with PE150 strategy. Library construction was performed according to manufacturer's instructions (10x Genomics) for 5' GEX, VDJ and CITEseq, V2 chemistry dual index