Annexin 5

Tumors were minced thoroughly and digested by 0.5 mg/ml Liberase TH (Sigma) for 30 minutes at 37°C. Dead cells were removed by Annexin V (STEMCELL technologies). scRNA-seq libraries were generated using the Chromium Single Cell 30 Reagent Kit v2 (10X Genomics). Cells were loaded onto the 10X Chromium Single Cell Platform (10X Genomics) at a concentration of 2,000 cells per μl (Single Cell 3’ library and Gel Bead Kit v.2) as described in the manufacturer’s protocol (10x User Guide, Revision B). Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT clean-up, complementary DNA amplification and library construction were all performed as per the manufacturer’s protocol. Individual sample quality was checked using a Bioanalyzer Tapestation (Agilent). Qubit was used for library quantification before pooling. The final library pool was sequenced on an Illumina NovaSeq6000 instrument using a S1 flow cell. Average cell recovery for Prkci^f/fPrkcz^f/f;Villin-Cre tumors was 104,954 cells with a total of 209,907 cells captured at a mean depth of 14,568 read per cell and 895 mean genes per cell.

Tumors were minced thoroughly and digested by 0.5 mg/ml Liberase TH (Sigma) for 30 minutes at 37°C. Dead cells were removed by Annexin V (STEMCELL technologies). scRNA-seq libraries were generated using the Chromium Single Cell 30 Reagent Kit v2 (10X Genomics). Cells were loaded onto the 10X Chromium Single Cell Platform (10X Genomics) at a concentration of 2,000 cells per μl (Single Cell 3′ library and Gel Bead Kit v.2) as described in the manufacturer’s protocol (10x User Guide, Revision B). On average, approximately 8,000 cells were loaded. Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT clean-up, complementary DNA amplification and library construction were all performed as per the manufacturer’s protocol. Individual sample quality was checked using a Bioanalyzer Tapestation (Agilent). Qubit was used for library quantification before pooling. The final library pool was sequenced on an Illumina NovaSeq6000 instrument using a S1 flow cell. Average cell recovery for the orthotopic in the Fsp1-cre mice was 23,576 cells, with a total of 47153 cells captured at a mean depth of 18712 reads per cell and 1224 mean genes per cell. Average cell recovery for the orthotopic in caecum was 15628 cells, with a total of 46886 cells captured at a mean depth of 18712 reads per cell and 1492 mean genes per cell.