Spatial transcriptomic profiling of decalcified murine musculoskeletal samples via Xenium Prime 5K

Area of Science:

• Molecular Biology
• Genomics
• Anatomy

Background:

• Spatial transcriptomics analysis of musculoskeletal tissues is challenging due to RNA degradation during harsh processing.
• Preserving RNA integrity through fixation and decalcification of mineralized tissues is a significant hurdle.

Purpose of the Study:

• To develop and validate a comprehensive sample processing pipeline for high-quality spatial transcriptomics of murine musculoskeletal tissues.
• To enable accurate transcript detection and anatomical context preservation in diverse tissue types.

Main Methods:

• A detailed protocol involving transcardial perfusion, fixation, decalcification, paraffin processing, and sample co-embedding was established.
• The Xenium Prime 5K platform from 10x Genomics was utilized for imaging-based spatial transcriptomics.
• Rigorous quality control measures were implemented at multiple processing stages.

Main Results:

• The pipeline successfully generated high-quality spatial transcriptomics data from intact knee joints, tibiae, and lumbar spines.
• High-quality transcript detection rates of 70%-91% were achieved across various tissues including synovium, bone, and dorsal root ganglion (DRG).
• Transcript counts per cell varied significantly by tissue type, with DRG neurons showing the highest yield.

Conclusions:

• The developed protocol effectively preserves RNA integrity and anatomical context in complex musculoskeletal tissues.
• This method facilitates high-resolution spatial transcriptomic profiling, enabling detailed cell type identification and gene expression analysis.
• The pipeline serves as a valuable tool for advancing research in musculoskeletal biology and disease.