GSM6380331: Sample1_GH2593_luc_live; Mus musculus; RNA-Seq
Primary PDAC tumors from KPCT and KPF; RosamTmG/+; Lgr5CreER mice were digested following method: tumors were finely chopped with scissors and incubated with digestion buffer containing collagenase IV (17104019, ThermoFisher Scientific, 0.1 U/ml ), Dispase (#354235, Corning, 0.6 U/ml), DNase I (#69182–3; Sigma Aldrich, 10 U/ml), and Soybean Trypsin Inhibitor (#T9003, Sigma Aldrich, 0.1 mg/ml) dissolved in HBSS with Mg2+ and Ca2+ (#14025076, Thermo Fisher Scientific) in gentleMACS C Tubes (Miltenyi Biotec) for 42 min at 37°C using the gentleMACS Octo Dissociator (#130–096-427, Miltenyi Biotech). After enzymatic dissociation, samples were washed with HBSS + Mg2+ and Ca2+ and filtered through a 100 μm cell strainer and spun at 300 g for 5 min at room temperature. Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 2% of heat-inactivated FBS in PBS) before being passed through a 40 μm strainer. At this point cells were used either for direct orthotopic implantations, for generation of 2D and 3D cells cell lines, or were prepared for FACS.  For FACS staining, cell suspensions were next blocked for 5 min at room temperature with rat anti-mouse CD16/CD32 with Fcblock (#553142, BD Biosciences) in FACS buffer, and incubated for 30min with a mix of four APC-conjugated antibodies against: CD45 (#559864, BD Biosciences, 1:500) + CD31 (#561814, BD Biosciences, 1:500) + CD11b (#561690, BD Biosciences, 1:500) and TER-119 (#561033, BD Biosciences, 1:500) and 300 nM DAPI as live-cell marker. Sorts were performed on a BD FACSAria sorter (Becton Dickinson) for either (CD45/CD31/CD11b/TER119)−/tdTomato+/DAPI− live cells (KPCT mice) and/or for (CD45/CD31/CD11b/TER119)−/tdTomato+/GFP+/DAPI− live cells (KPF; RosamTmG/+; Lgr5CreER mice) in lineage tracing experiments and as indicated in figures. Sorted cells were collected directly into S-MEM with 2% of heat-inactivated FBS for scRNA-seq or for scATAC-seq. If samples were hashed towards scRNA-seq sequencing, individual tumor cell suspension were incubated for 30 min with hashing antibodies (see table below) in addition to FACS antibodies. PDAC tumors were harvest, dissociated, and tumor cells isolated via FACS as described above. The scRNA-seq of FACS-sorted cell suspensions was performed on Chromium instrument (10X genomics) following the user guide manual for 3′ v3. Briefly, FACS-sorted cells were washed once with PBS containing 1% bovine serum albumin (BSA) and resuspended in PBS containing 1% BSA to a final concentration of 700–1,300 cells per μl. The viability of cells was above 80%, as confirmed with 0.2% (w/v) Trypan Blue staining (Countess II). Cells were captured in droplets. Following reverse transcription and cell barcoding in droplets, emulsions were broken and cDNA purified using Dynabeads MyOne SILANE followed by PCR amplification per manual instruction. Between 20,000 to 25,000 cells were targeted for each sample. Samples were multiplexed together on one lane of 10X Chromium following TotalSeq B cell hashing protocol  [49]. Final libraries were sequenced on Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles).