title: Single cell RNAseq of immune and stromal cells in murine lung cancer
design: Single cell RNAseq of immune and stromal cells in murine lung cancer
library_construction_protocol: Mice were sacrificed at age 6-10 weeks and tissue from lung or excised tumors on day 23 were disassembled and digested for further stromal cell or immune cell isolation and scRNA sequencing analysis. Tumor cell growth protocol: a murine lung cancer cell line Lewis lung carcinoma (LLC) line expressing H-2Db–restricted peptide 33-41 of the lymphocytic choriomeningitis virus (LCMV) glycoprotein (LLC-gp33) was used. LLC-gp33 cells were cultured in Iscove modified Dulbecco medium containing antibiotics and selection agents (Sigma-Aldrich). LLC-gp33 cells together with the fluorescent reporter protein tdTOMATO (tdTOM) were intravenously injected into Ccl19-EYFP mice. After 23 days lungs were harvested, subpleural tumor nodules were counted under the stereomicroscope and lungs were weighed. The mean weight of tumor-free lungs was subtracted for calculation of tumor weight per lung. Ccl19-EYFP+ cells from naïve lungs and established tumors on day 23 were isolated and further processed for single cell RNA-sequencing. Murine recombinant coronavirus vector (mCOV) treatment protocol: The recombinant murine coronavirus vector (mCOV) expressing the LCMV glycoprotein gp33 and the myeloid cell stimulating factor Flt3l (mCOV-Flt3l-gp33)(Perez-Shibayama et al, 2013) was used to boost anti-tumor T cell response. Mice were anaesthesized with isofluran and 5 x 104 plaque forming units (pfu) (in medium) were applied to the nose for inhalation. For enzymatic digestion of disaggregated lung tissue from tumors or excised tumors (day 23), 1 mg/ml Dispase (Roche), 1 mg/ml Collagenase Type II (Gibco) and 25 μg/ml DNaseI (AppliChem) in combination with gentleMACS-based mechanical disruption (Miltenyi Biotech) were used. After 30 minutes incubation at 37°C, cell suspensions were washed with PBS containing 0.5% FCS and 10 mmol/L EDTA. Enrichment of stromal cells was achieved by depleting hematopoietic and erythroid cells using MACS anti-CD45 and anti-Ter119 microbeads (Miltenyi Biotec). The flow-through was centrifuged and single cell suspension was stained for further flow cytometric analysis or cell sorting. The unbound single cell suspension was stained for cell sorting with a BD FACS Melody 2-way or 4-way cell sorter (BD Biosciences) and run on the 10x Chromium analyzer (10X Genomics). For the isolation of immune cells, tissues were gently smashed through a mesh using a syringe plunger and washed with PBS containing 0.5% FCS and 10 mmol/L EDTA before staining for flow cytometric analysis or cell sorting.  cDNA library generation was performed following the established commercial protocol for Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3/3.1 Chemistry.