GSM8231286: SL, scRNAseq; Mus musculus; RNA-Seq
The tumors developed in K14CreTg/+-Rosa26-Zeb2Tg/+-Trp53Fl/Fl mice were dissected, measured, finely chopped, and digested in 2 mg/ml collagenase A (Roche), 0.125 U/ml dispase II (Roche) and 1 µg/ml DNAse I (Roche) for 1 h at 37°C with gentle agitation. The digested tumor was passed through a 100-µm cell strainer, and the cells were washed in PBS containing 2% FCS. The cell suspension was stained DAPI for 1 h at 4°C. Tumor cells were sorted on a BD FACS ARIA II (BD bioscience) by forward and side scatter, doublet discrimination and DAPI exclusion. Tumor cells were selected by GFP positivity. The GFP+ tumor cells were cultured in DMEM supplemented with 10% FCS, penicillin/streptomycin, non-essential amino acids, and Na-pyruvate at 37°C under 5% CO2. Library prep was performed by the VIB Single Cell Core (VIB, Ghent-Leuven, Belgium). Sequencing libraries were loaded at 2.1pM loading concentration on a HiSeq4000 with custom sequencing metrics (single-indexed sequencing run, 28/8/0/98 cycles for R1/i7/i5/R2) (Illumina).