Single-cell RNA-sequencing (scRNA-seq) is a powerful technique that can barcode individual cells and thus used to obtain a gene expression profile for every individual cell within a tissue. This makes scRNA-seq an excellent method for characterizing rare cell populations such as stem cells. We describe how scRNA-seq can be utilized to examine limbal epithelial stem cell population as well as investigate the contribution of autophagy to the function of limbal epithelial stem cells. To accomplish this, we used the Beclin1 heterozygous (Beclin1 het) mouse, a well-established model of autophagy deficiency. We provide a protocol that we developed for the isolation of viable, single-cell suspensions of limbal/corneal tissues, as well as the analysis of scRNA-seq data.