Optimized protocol for processing murine tumor-bearing lung tissue for flow cytometry and single cell RNA-sequencing

NSCLC accounts for approximately 85% of lung cancers, and Kirsten rat sarcoma (KRAS) is the most commonly mutated gene in NSCLC. Although management of NSCLC has advanced markedly in recent years, its heterogeneity and plasticity remain incompletely understood. Single-cell RNA sequencing (scRNA-seq) has become an essential tool for dissecting cellular heterogeneity in complex tissues such as the lung. However, generating high-quality single-cell suspensions from tumor-bearing lung tissue is challenging, particularly during tumor formation when the tissue becomes fibrotic and heterogeneous. A key requirement for successful scRNA-seq is the preparation of viable single cells with minimal processing-induced stress or damage. Here, we present a detailed step-by-step protocol for isolating high-quality single-cell suspensions from both healthy and tumor-bearing mouse lung tissue in a KrasG12D; tdTomato reporter mouse model. The procedure includes tissue perfusion, controlled enzymatic digestion, gentle mechanical dissociation, red blood cell lysis, filtration, and viability assessment, and achieves >85% viable cells prior to scRNA-seq. Finally, the tdTomato+ tumor cells are efficiently isolated by FACS and can be detected as distinct clusters by scRNA-seq using this protocol. SUMMARYWe present a detailed protocol for isolating high-quality single-cell suspensions from both healthy and tumor-bearing murine lung tissue. This protocol can be applied to scRNA-seq, flow cytometry analysis, and primary cell isolation.