GSM5602252: CTV labeled memory T cells; Mus musculus; RNA-Seq
Spleens from OT-I and GFP;OT-I mice were harvested and CD8+ T cells were isolated using the Mouse CD8 T Lymphocyte Enrichment Set (BD Biosciences) using the manufacturers protocol. The obtained cells were mixed in a 1:1 ratio and transferred to 4 primary recipient C57BL/6J-Ly5.1 mice (1.5x106 T cells per recipient), and 24 hours later recipients were infected with 5,000-10,000 CFU Lm-OVA. 30 days post-infection spleens  were harvested from the primary recipients and were enriched for early memory CD8+ T cells using the Mouse CD8 T Lymphocyte Enrichment Set (BD Biosciences), replacing the manufacturers supplied antibody-cocktail with a mixture of anti-mouse CD19, CD20 and CD4 biotinylated antibodies (used 1:200 each, See Supplementary Table 7 for information on antibody clones). The enriched cell pool was subsequently stained with CellTraceä Violet (Thermofisher) and re-transferred into 4 infection-matched secondary C57BL/6J-Ly5.1 recipients. 74 after secondary transfer (104 days post-infection) spleens  were harvested from the secondary recipients and stained with anti-mouse KLRG1-PE, CD27-APC, and CD45.2-AF700 (See Supplementary Table 7 for information on antibody clones). Next, stained cell-pools were first enriched for transferred cells (i.e. CD45.2+) through FACS using the BD FACSAria™ Fusion Flow Cytometer (BD Biosciences), and subsequently sorted again to obtain 4 populations of TCM based on their GFP expression and CTV dilution: KLRG1-CD27+GFP+Division0-2, KLRG1-CD27+GFP+Division5+, KLRG1-CD27+GFP-Division0-2, KLRG1-CD27+GFP-Division5+. Each cell pool obtained by cell-sorting was barcode-labeled with distinct anti-mouse TotalSeq Hashtag antibodies (TotalSeq-A0301-0304, Biolegend), and subsequently pooled. Single-cell RNA isolation and library preparation was performed according to the manufacturer's protocol of the 10X Genomics Chromiumä Single Cell 3' kit, and the cDNA library was sequenced on a NextSeqTM 550 Sequencing System (Illumina). Feature-barcode matrices were generated using the Cell Ranger software of the 10X Genomics Chromium pipeline.