A Protocol for Harvesting Single-cell Suspension from Mouse Corneas

As the outermost layer of the eyeball, the cornea plays a critical role in maintaining ocular surface homeostasis and vision. Recent advances in single-cell RNA sequencing (scRNA-seq) have enabled high-resolution analysis of corneal cellular heterogeneity, yet the preparation of high-quality single-cell suspensions from corneal tissue remains challenging due to its dense and complex structure. In this study, we establish a robust protocol for generating single-cell suspensions from whole mouse corneas. The procedure involves mincing the corneas into fine fragments followed by sequential enzymatic digestion using collagenase A and trypsin. The resulting cell suspension was assessed for quality through multiple metrics: trypan blue staining was used to evaluate cell viability and count; immunofluorescence staining for Pancytokeratin and Keratocan enabled identification and quantification of major corneal cell types; and RNA integrity was verified using a bioanalyzer. This protocol consistently yields single-cell preparations with high cell numbers, excellent viability (≥ 94.6%), and a high proportion of single cells (96.3%), fulfilling the requirements compatible with demanding downstream applications, including scRNA-seq.