High-resolution profiling of osteocyte transcriptomes via single-nucleus RNA sequencing.

Area of Science:

• Molecular Biology
• Genomics
• Skeletal Biology

Background:

• High-throughput transcriptomics, including RNA sequencing (RNA-seq), revolutionized gene expression analysis.
• Single-cell RNA sequencing (scRNA-seq) revealed cellular heterogeneity but faced challenges in bone research, particularly with osteocyte isolation.
• Osteocyte isolation from mineralized bone matrix often results in low yields and artifacts, limiting transcriptomic studies.

Purpose of the Study:

• To develop and validate a single-nucleus RNA sequencing (snRNA-seq) protocol for efficient osteocyte transcriptomic profiling from bone tissue.
• To overcome limitations of scRNA-seq in bone research, specifically addressing low osteocyte yield and dissociation-induced artifacts.
• To generate a high-resolution gene expression map of osteocytes under physiological conditions.

Main Methods:

• Developed a novel snRNA-seq protocol optimized for snap-frozen, marrow-flushed long bones.
• Achieved high-yield recovery of osteocyte nuclei, minimizing dissociation bias and enhancing osteocyte representation.
• Applied the snRNA-seq method to mouse long bones, analyzing gene expression profiles.

Main Results:

• The snRNA-seq approach significantly increased osteocyte capture and transcriptomic fidelity (18.5%) compared to scRNA-seq (0.18%-6.64%).
• Identified a robust osteocyte transcriptomic signature, including the gene Sost, which is often undetected in scRNA-seq.
• Discovered 23 novel genes in osteocytes, including Tg, Kcnq5, and Rapgef4os1, potentially representing new regulators of osteocyte biology.

Conclusions:

• This study presents the first successful application of snRNA-seq for osteocyte analysis in bone tissue.
• The developed snRNA-seq protocol provides a valuable resource for studying osteocyte biology and skeletal disorders.
• The findings highlight potential novel regulators of osteocyte function and skeletal health.