GSM9069798: SPC KPI tumors; Mus musculus; RNA-Seq
Individual tumors were microdissected from ~30-week KP and ~40-week KPI animals (n = 5 tumors/animal; 2 animals/genotype). Microdissected tumors were dissociated using the Lung Dissociation Kit (Miltenyi Biotech, 130-095-927) according to the manufacturer's instructions. Dissociated cells were filtered using 70-μm and 30-μm strainers. CD31+ and TER-119+ cells were further removed using the EasySep Mouse Biotin Positive Selection Kit II (StemCell Technologies, 17665), followed by removal of dead cells using the EasySep Dead Cell Removal (Annexin V) Kit (StemCell Technologies, 17899). scRNA-seq was performed on purified suspensions of epithelial and immune cells using the Chromium Next GEM Automated Single Cell 3' Library & Gel Bead Kit v3.1 (10X Genomics, 1000147) according to the manufacturer's instructions. Briefly, cell number and viability were determined using 0.2% (w/v) Trypan Blue staining. 10,000 cells were then loaded onto the Chromium Connect for an automated process of GEM generation, post GEM-RT cleanup, cDNA amplification, and 3' gene expression library construction. Single-cell libraries were then sequenced on the Illumina NovaSeq S4 by the Mayo Clinic Genome Analysis Core.