Evaluation of the digestion protocol of mouse neonatal epidermis for single-cell RNA sequencing.

Abstract

The skin is primarily composed of keratinocytes and forms an effective barrier between the organism and external environment. Neonatal skin analysis is essential for understanding developmental processes and rare skin diseases. However, efficient single-cell dissociation methods for the neonatal mouse epidermis remain underexplored. Here, three enzymes (Trypsin, TrypLE, and Liberase) used for tissue dissociation were compared to optimize single-cell RNA sequencing (scRNA-seq) of the mouse neonatal epidermis. scRNA-seq revealed distinct differences in cell recovery between the enzymes, with Liberase enriching suprabasal keratinocytes and Trypsin/TrypLE favoring basal keratinocytes. Although all enzymes produced comparable data quality, the observed bias in cell population recovery highlights the significant impact of dissociation protocols on the scRNA-seq results. These findings highlight the importance and optimal selection of enzymes for the analysis of unbiased neonatal epidermis.