title: GSM7136074: DAA, Early-tx, subject 3, scRNAseq; Homo sapiens; RNA-Seq
library_construction_protocol: PBMCs were thawed at room temperature in RPMI media and centrifuged at 350xg for 10min at 4˚C. Cells were washed with Washing Buffer (PBS, pH 7.4 + 5% FBS), centrifuged, and the cell pellet was resuspended in Stain Buffer (PBS, pH 7.4 + 5% FBS + 0.1g/L Herring sperm DNA). Then, we added 0.2 µl 100µM d-Biotin (Avidity LLC, Aurora, CO) per included dextramer and added 2µl of desired dCODE™ Dextramer®-PE reagents (Immudex, Denmark), including the negative control for allele and random dextramers. After 10 min of incubation in the dark, we added the antibody cocktail for CD8+ T cell staining (CD4-APC, CD3-FITC, CD8-V500, and CD56-V450) and incubated for 30 min at 4˚C in the dark. We sorted the CD3+ CD8+ Dextramer+ cells using BD FACSAria™ Fusion into 90% FBS solution at 4˚C. Sorted cells were centrifuged and resuspended in 50µl of PBS with 0.04% BSA for scRNAseq partitioning. Library was performed according to the manufacter's instructions (single cell 5' v1.1 or v2 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.