Single-Cell RNA Sequencing of Lung Tissue in a Rat Model of Acute Respiratory Distress Syndrome

Acute Respiratory Distress Syndrome (ARDS) is a severe inflammatory lung condition with high mortality, characterized by dysregulated immune responses and alveolar damage, yet its cellular heterogeneity remains incompletely understood. Single-cell RNA sequencing (scRNA-seq) offers unprecedented resolution to dissect cell-type-specific mechanisms, but its application in ARDS is hampered by technical variability. Here, we present a standardized protocol for scRNA-seq analysis of lung tissue in a lipopolysaccharide (LPS)-induced rat ARDS model. This method includes optimized tissue dissociation, rigorous quality control, doublet removal, batch effect correction, and comprehensive bioinformatic pipelines for clustering, annotation, and differential expression analysis. Our approach reliably identifies 21 distinct cell populations and reveals six macrophage/monocyte subpopulations with unique transcriptional signatures in ARDS. Subcluster-specific differential gene expression and functional enrichment analysis further identified key genes and pathways underlying ARDS pathogenesis. This reproducible workflow enables in-depth characterization of cellular diversity, inflammatory pathways, and candidate therapeutic targets, providing a robust foundation for mechanistic and translational studies in ARDS.