Expansion sequencing: Spatially precise in situ transcriptomics in intact biological systems.

Area of Science:

• Molecular Biology
• Neuroscience
• Genomics

Background:

• Current multiplexed RNA imaging lacks nanoscale resolution, limiting transcript localization to subcellular compartments.
• Accurate spatial transcriptomics is crucial for understanding cellular function and disease.

Purpose of the Study:

• To adapt expansion microscopy for in situ RNA sequencing with nanoscale resolution.
• To develop a method for highly multiplexed, long-read RNA sequencing applicable from subcellular to system scales.

Main Methods:

• Adapted expansion microscopy for untargeted and targeted in situ RNA sequencing.
• Applied expansion sequencing (ExSeq) to mouse brain, hippocampus, and human metastatic breast cancer biopsy.

Main Results:

• Untargeted ExSeq provided readout of thousands of genes, including splice variants, in the mouse brain.
• Targeted ExSeq generated nanoscale RNA maps in hippocampal neurons, revealing dendritic and spine-level transcript localization.
• ExSeq identified cell-type-specific and layer-specific RNA patterns in the visual cortex and mapped tumor and immune cells in human cancer biopsies.

Conclusions:

• Expansion sequencing (ExSeq) overcomes resolution limitations in RNA imaging.
• ExSeq enables highly multiplexed mapping of RNA from nanoscale to system scale.
• This technique offers unprecedented insights into spatial transcriptomics across diverse biological systems.