Single cell RNAseq of immune and stromal cells in murine lung cancer
Single cell RNAseq of immune and stromal cells in murine lung cancer
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. To validate the differentiation process of fibroblasts in murine lung cancer, fatemapping experiments were performed using the inducible tetracycline transactivase (Tta)-based Ccl19-Tta mouse model in combination with single-color R26R-EYFP (Ccl19-iEYFP) mice. Ccl19-iEYFP mice were treated with doxycycline between day 15 and 23, enabling progeny of day 15 progenitors to be analysed on day 23. Then, single cell RNA-sequencing of fate mapped Ccl19-iEYFP+ cells was performed. 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.