Single-cell sequecing of LCMV-specific WT and HIF-1a-deficient CD8 T cells isolated from LCMV-Clone13 infected host mice

To determine the role of hypoxia-inducible factor 1 alpha (HIF-1a) in exhausted T cells, we crossed Hif1a-fl/fl Cd4-Cre mice to tdTomato+ P14 animals and adoptively co-transferred equal numbers of WT and HIF-1a-deficient P14 CD8+ T cells into chronically infected WT host mice. 21 days post infection, we retrieved GFP+ (WT) and tdTomato+ (HIF-1a-deficient) donor P14 T cells by FACS sorting and subjected CITE-seq antibody-barcoded WT and HIF-1a-deficient CD8+ P14 T cells to multiplexed scRNA sequencing analysis. Overall design: CD8+ tdTomato+ (HIF-1a-deficient) and CD8+ tdTomato+ GFP+ (WT) P14 T cells were FACS-sorted from the spleen and LNs of chronically infected mice using a FACSAria III cell sorter (BD Biosciences). To multiplex different samples for scRNA sequencing, single-cell suspensions after FACS sorting were labelled with different TotalSeqA antibodies (Biolegend). Each sample was labelled with one specific hashtag (1:400 dilution) and CITE-seq antibodies (1:800 dilution, all Biolegend). After labelling for 30 min at 4 °C, samples were washed three times and multiplexed in equal cell numbers at a density of 106 cells/ml.