GSM7885651: YUMM3_3-ActA_GFP_2; Mus musculus; RNA-Seq
YUMM3.3-Ctrl.GFP and -ꞵA.GFP tumors were dissected, minced using rounded scissors, and digested in Dnase-I (0.02mg/mL, Sigma) and collagenase (1mg/mL, Sigma) in RPMI using a gentleMACS Octo Dissociator (Miltenyi). In total, we collected six Ctrl and six ꞵA tumors. Two tumors of each genotype were pooled for each biological replicate based on tumor volumes to have comparable starting amounts for each replicate. To minimize the time that cells spent on the ice, samples were randomized, and live single-cell sorted on two machines (Becton Dickinson FACSAria II and ACSAria Fusion) into GFPhi tumor cells and GFPint stromal populations. For dead staining, Propidium Iodide Solution (421301, Biolegend) was added to the cells immediately before sorting. Immediately after sorting, cells were counted, centrifuged at 300g for 5 min at RT, and resuspended at a density of 1000 cells/μl in DMEM supplemented with 10% filtered FBS. GFPhi fractions were added back to GFPint cells to account for 10% of the final volume Cell concentrations and quality were assessed using trypan blue staining. Samples contained less than 5% dead cells and 3-5% doublets. Single-cell RNA sequences were prepared using 10x Genomics Chromium v3.1 kit according to the manufactur's instruction