Nfe2l2-knockout mouse model recapitulates key pathological features of keratoconus

Fresh corneas from WT and Nfe2l2-KO mice (n = 5/group, 4-month-old) were dissociated with papain (20 U/mL) + DNase I for 15–20 min at 37 °C, quenched with ovomucoid, filtered (40 µm), and processed on a droplet-based scRNA-seq platform. Library preparation and sequencing were outsourced to Singleron Biotechnologies (Nanjing, China). Raw reads were aligned to mm10 with STAR/Drop-seq Tools; ambient RNA was corrected with SoupX. In Seurat v5, low/high feature outliers and high-mitochondrial cells were removed, doublets were filtered (DoubletFinder), SCTransform normalization and integration were applied, clusters were identified by SNN/UMAP, and annotated with canonical markers. Differential expression used Wilcoxon with BH FDR < 0.05. Functional enrichment of DEGs was performed with Metascape. Cell–cell communication was inferred with CellChat v1.6 (MouseDB; 1,000 permutations; FDR < 0.05) and summarized at pathway level.