title: GSM6704043: TDLN-E-B16-WTISO-CD8; Mus musculus; RNA-Seq
library_construction_protocol: scRNA-seq on tumor-infiltrating CD8+ TILs:Tcf7fl/fl (wildtype) and E8i-CRE+ x Tcf7fl/fl (TCF1 KO) mice were injected with 3.75x105 B16-OVA or 1x106 MC38-OVA cells subcutaneously in the right flank. On day 6 and 9, mice were randomized and treated intraperitoneally with a combination of anti-PD-L1 (10F.9G2, 200g per mouse) and anti-Tim-3 (RMT3-23, 200g per mouse) antibodies or control immunoglobulins (Rat IgG2a and Rat IgG2b). Two (for MC38-OVA tumors) or three (for B16-OVA tumors) days after the second dose, tumors were harvested (n=3-5 per group) dissociated to single cell suspensions prior to fluorescence-activated cell sorting. An equal amount of CD45+ TCRb+ CD8+ T cells were sorted from each mouse (10,000 cells/mouse) within each experimental group and then pooled together prior to scRNA sequencing. scRNA-seq on adoptively transferred OTI T cells from the TDLN: CD45.1 C57Bl/6 were injected with 3.75x105 B16-OVA or 1x106 MC38-OVA cells subcutaneously in the right flank. On day 5 post tumor implantation, mice were randomized by tumor size and given 1x106 TCF7 KO or WT OT-1 naïve T cells resuspended in PBS via tail vein injections. Mice were then treated on day 6 post tumor injection with one dose of ICB (PDL1 + Tim3, 200ug each) or control antibodies (Rat IgG2a and Rat IgG2b). On day 8 post tumor injection the inguinal tumor-draining lymph nodes (TDLN) were dissected and dissociated to single cell suspensions by passing tissue through a 70M filter prior to centrifugation and fluorescence-activated cell sorting. CD45.2+CD8+TCRVa2+ OTI T cells were purified from the TDLN of n=4-5 mice per group. For the generation of single cell transcriptomes, 10^3 to 16x10^3 -  target cells from each sorted population were run using the Chromium Controller (10x Genomics) using Chromium Next GEM Single Cell 3' Reagent Kits (10x Genomics). The libraries generated were then run on the NextSeq 550 Sequencing System (Illumina) using NextSeq 500/550 High Output v2.5 (150 cycles) Kit (Illumina).