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

Area of Science:

• Molecular Biology
• Genomics
• Anatomy

Background:

• Spatial transcriptomics offers insights into cellular function within anatomical context.
• Preserving RNA integrity in mineralized musculoskeletal tissues for transcriptomics is challenging.
• Existing methods struggle with harsh fixation and decalcification steps.

Purpose of the Study:

• To develop and validate a comprehensive pipeline for high-quality spatial transcriptomics in murine musculoskeletal tissues.
• To enable transcript detection across diverse tissue types including mineralized and soft tissues.
• To facilitate simultaneous sectioning of multiple samples for anatomical consistency.

Main Methods:

• Transcardiac perfusion, fixation, decalcification, and paraffin processing.
• Sample co-embedding strategy for simultaneous sectioning onto spatial transcriptomics slides.
• Utilized the 10x Genomics Xenium Prime 5K platform for imaging-based spatial transcriptomics.

Main Results:

• Achieved 70-91% high-quality transcripts across various musculoskeletal tissues (synovium, bone, cartilage, muscle, etc.).
• Detected varying transcript numbers per cell, from ~13 in cortical bone to ~1100 in dorsal root ganglion neurons.
• Confirmed successful identification of key cell types using canonical cell markers.

Conclusions:

• The developed pipeline successfully generates high-quality spatial transcriptomics data from challenging murine musculoskeletal samples.
• This protocol preserves anatomical context and RNA integrity, enabling detailed cellular and tissue-level analysis.
• The method is a valuable tool for advancing research in musculoskeletal biology and disease.