title: GSM6568079: Sample1_High, scRNAseq; Mus musculus; RNA-Seq
library_name: GSM6568079
library_construction_protocol: CT26 tumors were harvested from BALB/c mice, minced using a razor blade, and treated for 25 min at 37°C with 0.1 mg/ml Liberase (Research Grade, Dispase Low) in 5mL serum-free RPMI 1640 medium according to the manufacturer's instructions (Roche Life Science). Tumors were filtered through a 100-μm cell strainer and washed in complete medium [RPMI with L-glutamine, 10% FBS, 100 U/ml each penicillin and streptomycin, 1 mmol sodium pyruvate, 10 mmol HEPES, and 1× MEM nonessential amino acids]. Splenocytes were harvested from mice, mechanically dissociated, filtered through a 100 μm cell strainer, treated for ∼5 min with ammonium chloride-potassium lysis buffer, and washed in complete medium. For scRNAseq experiments, tumors from 15 mice were pooled together to make a single sample. Before FACS, CD8+ T cells were enriched using a Mouse CD8 Negative Selection Kit (Stemcell Technologies) according to the manufacturer's instructions. CITE-seq antibodies were added during the final 30 minutes of tetramer staining and added to samples before staining with fluorophore conjugated antibodies for flow sorting. After the 30-minute incubation samples were washed twice with FACS buffer. CD8-enriched TIL were sorted based on tetramer binding into Tet-High, Tet-Low and Tet-Negative groups then were immediately processed for scRNAseq. Using the Biolegend TotalSeq-A antibodies and protocol along with the Chromium Single Cell 3' Reagent Kits v3, and CITE-seq were performed. Cells were received in the Genomics Core, viability was assessed using TC20 (Biorad), cells were diluted to an appropriate volume to target 5000 cells. The samples then went onto the 10x Chromium machine for GEM generation and barcoding. Briefly, 10x Barcoded Gel Beads are mixed with the surface protein labeled cells in a master mix and partitioning oil to obtain single cell GEMs. Gel beads are dissolved allowing for incubation of both the poly A RNA and cell surface proteins within each GEM resulting in full length cDNA and DNA from the cell surface protein. GEMs are then cleaned following step 2.1 in the Chromium Single Cell 3' Reagent Kits v3 protocol. Once done with the clean-up, Protocol – TotalSeq-A Antibodies with 10x Single Cell 3' Reagent Kit v3 3.1 was followed at Step II. cDNA amplification mix was prepared according to this protocol to allow for Antibody Derived Tag (ADT) libraries. The libraries were finished with the Protocol – TotalSeq-A Antibodies with 10x Single Cell 3' Reagent Kit v3 3.1. Sequencing was performed at the University of Colorado Genomics Shared Resource lab on the Illumina NovaSeq 6000.