Comparative Transcriptomic Profiling of Corneal Compartments Using Single-Cell and Single-Nucleus Sequencing.

Purpose: Single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA-sequencing (snRNA-seq) offer complementary advantages for corneal transcriptomics study. However, variations in cell-type distribution across platforms and the technical challenges in isolating corneal cells may lead to misinterpretation of biological conclusions. This study aimed to establish cell-type-specific recommendations for corneal transcriptomics. Methods: We systematically evaluated scRNA-seq and snRNA-seq performance using matched human and mouse corneal datasets. The expression of Col8a1 and Kdr was measured by immunofluorescence. Results: Compared to snRNA-seq, scRNA-seq enabled more accurate prediction of differentiation trajectory in corneal epithelial cells. Moreover, scRNA-seq captured a higher proportion of stromal keratocytes and showed elevated expression of stromal disease-associated genes, whereas snRNA-seq more effectively identified and characterized corneal endothelial cells. Conclusions: Collectively, our findings provide an objective evaluation of scRNA-seq and snRNA-seq performance across corneal cell types, offering practical guidance for selecting optimal sequencing strategies based on specific research requirements.