Mouse Tail-Skin Dissociation and Preparation of Live Single-Cell Suspension for Downstream Analysis of Melanocytes

Abstract

Isolating high-quality viable single cells from mouse tail skin, a well-established model for studying skin cells and melanoma pathogenesis, is challenging due to the presence of dense connective tissue and hair follicles. Single-cell RNA sequencing (scRNA-seq) is a powerful tool for studying skin cell heterogeneity. However, the lack of a robust protocol for the efficient generation of highly viable single-cell suspension from mouse tail skin has limited its application for studying melanocyte-interacting cells and characterizing the melanocyte niche. We developed a robust protocol for generating highly viable single-cell suspensions from mouse tail skin, facilitating single-cell transcriptomic profiling of keratinocytes, melanocytes, and fibroblasts. We demonstrate the successful isolation of melanocytes and other melanocyte-interacting cells using our protocol and a proof-of-concept scRNA-seq study for interrogating the melanocyte niche. Our protocol employs a two-stage enzyme dissociation step, followed by debris removal and subsequent live cell enrichment, to obtain a single-cell suspension with high cell viability. This straightforward protocol enables the isolation of viable single cells from mouse tail skin for downstream scRNA-seq studies. Further, this approach allows comprehensive analysis of the melanocyte niche and melanocyte-interacting cells, potentially aiding in identifying the melanoma cell of origin.