GSM7139810: Pancreatic tumor, Tgm2-edited, rep1; Mus musculus; RNA-Seq
Orthotopic tumors were collected, minced, and incubated at 37°C in digestion buffer [RPMI (Gibco), 2% FBS, 0.2 mg/mL Collagenase P (Roche), 0.2 mg/mL Dispase (Gibco), and 0.1 mg/mL DNase I (Roche)] with low agitation (300 rpm) for 45 min, vortexing every 15 min. Released cells were collected from the supernatant and quenched with cold flow cytometry buffer (PBS, 2% FBS, 20 mM HEPES). The remaining tumor pieces underwent a second round of digestion. Released cells from the two rounds were combined, filtered through 70-um mesh, and concentrated by centrifugation (300 rcf, 5 minutes, 4°C). The single cell suspension was then subjected to ACK lysis and a series of negative selection for removal of dead cells (Miltenyi Dead Cell Removal Kit, cat No. 130-090-101), immune cells (Miltenyi CD45 microbeads, cat No. 130-052-301), and epithelial cells (Miltenyi EPCAM microbeads cat No. 130-105-958). Remaining cells were used for single cell sequencing on the 10X Genomics platform. Libraries were prepared with the Chromium Single Cell 3' Reagent Kit (v3.1), following the manufacturer's instructions.