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.