GSM5827719: Spleen, Tregs (HTO); Mus musculus; OTHER
Splenocytes were harvested from Foxp3Cre-YFP or Ifngr1L/LFoxp3Cre-YFP mice that remained naïve or were infected with Cl. 13. for 16d. T cells were enriched by negative selection. Briefly, single cell splenic suspensions were incubated with a cocktail of the following biotinylated antibodies at 4oC for 15 min: CD11b (M1/70), CD11c (N418), CD19 (6D5), CD45R/B220 (RA3-6B2), TCRgd (GL3), PanNK (DX5), CD105 (MJ7/18), CD16/32 (93) and Ter119 (TER-119). Non-T cells were removed by incubating the suspension with streptavidin-coated magnetic beads at 4oC for 15 min, followed by magnetic separation. The unbound pure T cells were incubated with pooled MHC class I tetramer (APC) (GP33, GP276, NP396) and then stained for viability using Live/Dead Fixable eFluor 780. T cells from individual mice were identified via labeling with a uniquely barcoded TotalSeq-C antibodies towards pan-CD45 and MHC at 1:100 according to manufacturer's protocol during surface staining (Biolegend). Tregs and Tetramer+ CD8+ T cells purified via fluorescently activated cell sorting using a BD Aria. Live cells were spun down, resuspended in 0.04% PBS-BSA (Fisher #MT20031CV and Sigma #A9418-100G), and counted to determine a cell stock concentration.A cell suspension volume was calculated to capture approximately 7,000 cells per sub-population. Cells were added to a Master Mix containing Reagent mix, RT Primers, Additive A, and RT Enzyme Mix to create a reaction for Tregs and one for Tetramer+ CD8+ T cells. These reactions were then loaded into separate lanes on Chromium Chip A and run through the 10X Genomics Chromium Controller. All libraries were sequenced on a NovaSeq™ 6000 S2 kit for 150 cycles at the UPMC Genome Core. Each Gene Expression library was sequenced at a depth of at least 20,000 reads/cell. Each Feature Barcode and TCR library was sequenced at a minimum depth of 5,000 reads/cell.