GSM7506140: 4virus VDJ, 3 donors, scRNAseq; Homo sapiens; RNA-Seq
For single-cell RNA-seq using 10X genomics, PBMCs from 10 donors that showed highest response to peptide stimulation in preliminary experiments, were stimulated with pp65 and IE-1 PepTivators for 6 and 24 hrs. The stimulated cells from each donor were washed and individually stained with Cell-hashtag (HTO:Hash Tag Oligo; D1-D10) TotalSeq C antibody (Biolegend) (0.5 µg/condition) as per manufacturer's recommendations for 30 minutes on ice. The stained and washed PBMCs from all 10 donors were then pooled and stained with oligo-tagged TotalSeq C antibody for cell surface proteins for T cell memory markers (CD45RA, CCR7, CD95 and IL7Rα) (Biolegend) (0.5 µg/condition) for 30 minutes on ice. Following washing, the pooled cells were further stained with fluorescence-conjugated surface antibodies to enable FACS sorting of antigen-specific cells. Cells were sorted using FACSAria Fusion Cell Sorter (Becton Dickinson) in LoBind 1.5 mL microcentrifuge (Eppendorf) tubes with 50% FBS supplemented with recombinant RNase inhibitor (1:100, Takara). Cells were then centrifuged for 5 minutes (500 g at 4 C) and the supernatant was carefully removed leaving 5 to 10 µl. 25 µl of resuspension buffer (0.22 µm filtered ice-cold PBS supplemented with ultra-pure bovine serum albumin; 0.04 %, Sigma-Aldrich) was added to the tube and the pellet was gently but thoroughly resuspended. Following careful mixing, 33 µl of the cell suspension was transferred to a PCR-tube for processing as per the manufacturer's instructions (10x Genomics). scRNA-Seq with 10x Genomics 5' v1.1; The sorted antigen-specific cells from various memory compartments were pooled in desired ratios and used for single-cell RNA-Seq and TCR-Seq assay using 10X Genomics kit, 5ʹ V(D)J with gene expression and cell surface protein expression v1.1 as per the manufacturer's recommendations. Initial amplification of cDNA libraries and final libraries were performed for 15 cycles and 14 cycles respectively for gene expression library. V(D)J and cell surface protein libraries were generated corresponding to each 5ʹ gene expression library using 9 cycles of amplification. Libraries were sequenced on Illumina NovaSeq6000 sequencing platform for PE150 reads.