GSM8155269: pooledCells_scGEX_T1; 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). 5x10^6 PBMCs per donor and timepoint were recovered and first stained with a cocktail of 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 andwere later distinguished by individual CD45 color-barcoding at the sorter and by individual TotalSeq-C anti-human hashtag antibodies in the scRNAseq dataset. Meanwhile, a dextramer cocktail containing 16 different dextramers was prepared. For this cocktail, 8 µL per 40x10^6 cells of dCODE dextramers® (immudex) and 100 µM d-biotin (1/10 of total dextramer volume) were pre-mixed in FACS-buffer (PBS + 0.5% BSA) to block free binding sites. The pooled samples (40x10^6 cells) were stained with 400 µl of this dextramer cocktail for 30 min on ice. Single, live, CD19-, CD56-, CD8+, dextramer-positive lymphocytes were sorted in previously FCS-coated 1.5 mL tubes filled with FACS-buffer. 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 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