Dnase 1

Micro-dissected lung tumors were dissociated with collagenase IV, dispase, and trypsin at 37°C for 30 minutes as previously described^{118 (link)}. The digestion buffer was then neutralized with cold L-15 media (Thermo Fisher Scientific: 21083027) containing 5% FBS (Gemini Bio) and DNaseI (Sigma-Aldrich: DN25). Dissociated cells were treated with ACK Lysis Buffer (Thermo Fisher Scientific: A1049201) and resuspended in PBS containing 2 mM EDTA (Promega: V4233), 2% FBS, and DNase I. For the isolation of neoplastic cells, dissociated cells were stained with DAPI and antibodies against CD45 (BioLegend: 103112; 1:800 dilution), CD31 (BioLegend: 102402; 1:800 dilution), F4/80 (BioLegend: 123116; 1:800 dilution), and Ter119 (BioLegend: 116212; 1:800 dilution) to exclude hematopoietic and endothelial cells. FACSAria™ sorters (BD Biosciences) were used for cell sorting. For sorting of total cells within tumors for single-cell RNA-seq, dissociated cells were stained with DAPI only to exclude dead cells. Representative gating scheme included in Supplementary Fig. 20a.

Excised ear tissue was chopped and incubated with 1mg/mL Collagenase IV (Life Technologies, Carlsbad, CA) and 4U DNAse I (Thermo Fischer Scientific, Pittburgh, PA) for 30 minutes at (37 °C) before inactivating with 200 μL foetal bovine serum. Lysates were passed through a 70 μm strainer and cells pelleted by centrifugation with an additional 4U DNAse I Cells were incubated with purified anti-CD16/32 (Clone 93, Biolegend, San Diego, CA) for 15 minutes at 4 °C before washing and staining with a cocktail of fluorescently-conjugated anti-mouse monoclonal antibodies for 30 minutes at 4 °C: CD45.2 PercP Cy5.5 (Clone 104), CD11c PECy7 (N418), Ly6C APC (HK1.4RUO), (all from Affymetrix, San Diego, CA), CD11b Brilliant Violet 605 (M1/70), F4/80 Brilliant Violet 421 (BM8) (Biolegend, San Diego, CA) and Ly6G FITC (1A8), Siglec F PE (E50-2440) (Becton Dickinson, Franklin Lakes, NJ Data were acquired on a BD LSR II flow cytometer and analysed using Flowjo v9 (TreeStar, Ashland, OR)). DRAQ 7 (Biostatus, Shepshed, UK) was used to exclude dead cells before analysis. Doublets and debris were removed based on forward and side scatter properties before gating.

Uteri were removed from mice, cut longitudinally, and then cut into ~0.25 cm sections and placed in 10× Gentle Collagenase/Hyaluronidase (Stem Cell Technologies #07919) diluted 1:10 in DMEM F12 (Gibco #12634-010) for 4 h at 37°C with intermittent vortexing. Digested uteri were centrifuged for 5 min at 450g, resuspended in ammonium chloride (Stem Cell Technologies #07800) to lyse the red blood cells, and washed with PBS. Digested uteri were centrifuged again, resuspend in Trypsin EDTA (Stem Cell Technologies #07901), triturated prior to adding HBSS + 2% FBS (HF), recentrifuged and resuspended in Dispase with 1 mg/ml DNaseI (Stem Cell Technologies #07913 & #07900) by trituration. Cells were resuspended in HF and 0.1 mg/ml DNaseI and incubated with EpCAM (Biolegend #118217), CD11b (TONBO Biosciences #20-0112) and Thy1 (Biolegend #105325) antibodies for 30 min at 4°C in the dark. After staining cells were washed two times in HF and resuspended in HF with DNaseI with DAPI. Data was acquired using Beckman Coulter MoFlow Astrios and at least 500 000 events were collected in the live, single-cell gates for experimental tubes and 200 000 events for control tubes. Fluorescence minus one controls were used to set gates and single color tubes for both antibodies and endogenous fluorescence were used for compensation. Data were analyzed using Beckman Coulter Kaluza Analysis software.