NextSeq 500 paired end sequencing; Single cell RNA-seq of human CD8+ T cells binding the IAV HLA-A24/PB1 498-505 tetramer
Single cell RNA-seq of human CD8+ T cells binding the IAV HLA-A24/PB1 498-505 tetramer
PBMC from for donors were FcR blocked and stained with PE-conjugated HLA-A24:02 IAV PB1 498-505 tetramer. Cell were enriched using tetramer-associated-magnetic-enrichment and PE+CD3+CD8+ single cell sorted into a 96-well plate on BD Aria III. Single tetramer+ cells were sorted into 96-well plates containing 0.5 μl dNTP mix (10 mM), 0.5 μl oligo-dT pri- mer (5 μM), and 1 μl lysis buffer (prepared by adding 1 μl RNase inhibitor to 19 μl Triton X-100 solution, 0.2% v/v). Libraries were prepared using the Smart-seq2 protocol with the following modifications. cDNA synthesis and pre-amplification reaction volumes were halved to 5 and 12.5 μl, respectively. The final concentration of the ERCC RNA Spike-In Mix in the reverse-transcription step was 1:400 million.