GSM9476900: Vaccine bioreps 1-3, Naive bioreps 1-3; Mus musculus; RNA-Seq
To generate effector H2-Kb:VVLSFELL-specific CD8 T cells for sequencing, C57BL/6 mice were immunized intravenously with 150 μg RBD protein, 40 μg poly(I:C) and 40 μg anti-CD40. Seven days later, splenocytes from three mice were incubated with H2-Kb:VVLSFELL-APC tetramer (NIH Tetramer Core) in RPMI 1640 containing 10% FBS, 10 mM HEPES, 0.1 mM beta mercaptoethanol, 0.1 mM non-essential amino acids, 0.1 mM sodium pyruvate, 2 mM l-glutamine, and penicillin-streptomycin (complete media), for 30 mins at 37°C. Cells were labeled with anti-APC microbeads (Miltenyi) and enriched via magnetic column separation. Tetramer-enriched cells were stained and flow-sorted as live CD44highB220−CD8+ dual tetramer+ (APC/BV421) live cells using a BDFACSAria (BD Biosciences). As a comparator, splenocytes from 3 unmanipulated mice magnetically enriched for CD8 T cells (Bio-Legend) and sorted as live CD44lowB220−CD8+ cells. Post-sort purities exceeded 99% for both populations. Each sorted population was stained with a unique oligo-labeled sample tag (BD Biosciences) before library preparation. Whole transcriptome (WTA) and V(D)J libraries were prepared using the BD Rhapsody Single-Cell Analysis System with the “early-access” mouse VDJ reagents now sold as the BD Rhapsody Mouse TCR/BCR Next Amplification Kit following the manufacturer's protocol. Libraries pooled in equimolar ratios and sequenced on an Illumina NextSeq 500-series platform by BD Biosciences.