Protocol for the enrichment of endosteal and periosteal mesenchymal cells from murine bone for single-cell transcriptome analysis.

Bone comprises diverse cell populations essential for skeletal development, remodeling, and homeostasis. Current single-cell isolation methods often use bulk digestion that can obscure distinctions between periosteal and endosteal compartments. Here, we present an optimized murine bone single-cell RNA sequencing (scRNA-seq) workflow using Liberase™ digestion, EDTA decalcification, and sequential depletion of hematopoietic and endothelial cells to improve yield and viability. We also provide a basic Seurat (R) pipeline for data processing and visualization. For complete details on the use and execution of this protocol, please refer to Nookaew et al.1.