GSM9452682: MC38, Tumor Cells, Colon adenocarincoma; Mus musculus; RNA-Seq
MC38-SIY colon carcinoma and B16-SIY melanoma cell lines (Gajewski Laboratory, University of Chicago) were cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 1× HEPES at 37 °C, 5% CO2. Cell lines were routinely tested for mycoplasma. Tumor cells were detached with 0.25% trypsin-EDTA, washed, and 2 × 106 cells in 100 µL PBS were injected subcutaneously into the flank. Tumors were harvested 14 days post-implantation. Tumors were minced and digested for 20 min at 37 °C in RPMI containing 250 µg/mL Liberase (Sigma-Aldrich) and 50 µg/mL DNase I (Sigma-Aldrich), then mashed through a 70 µm strainer. Tumors of the same type were pooled, washed three times in chilled FACS buffer (PBS + 1% FBS + 2 mM EDTA), blocked with anti-CD16/32 (clone 93, BioLegend; 1:100), and stained with Fixable Viability Dye eFluor 780 (eBioscience; 1:2000) and fluorophore-conjugated antibodies. Live CD45+ cells were sorted on a BD FACSAria III into RPMI + 10% FBS. Sorted cells were resuspended in PBS + 0.04% BSA at ~1,000 cells/µL. Single-cell 3′ gene expression libraries (no CITE-seq or TCR sequencing) were generated using Chromium Single Cell 3′ Reagent Kits v2 (10x Genomics) and sequenced on an Illumina HiSeq 2000.