Spatial isoform sequencing at sub-micrometer single-cell resolution reveals novel patterns of spatial isoform...

Area of Science:

• Molecular Biology
• Neuroscience
• Genomics

Background:

• Current spatial long-read technologies often lack single-cell resolution, hindering the distinction between cell-type abundance and cell-type-specific isoform variation.
• Understanding spatial gene expression at the isoform level is crucial for deciphering cellular heterogeneity and function within complex tissues like the brain.

Purpose of the Study:

• To develop and validate a high-resolution spatial transcriptomics method (Spl-ISO-Seq2) capable of resolving isoform-level spatial variation within individual cell types.
• To investigate spatial isoform patterns in the adult mouse brain and differentiate between cell-type-specific isoform variability and regional cell-type abundance differences.

Main Methods:

• Development of Spl-ISO-Seq2 with 220nm spot size and 500nm resolution.
• Application to adult mouse brain tissue.
• Analysis using accompanying software packages Spl-IsoQuant-2 and Spl-IsoFind.
• Comparison of differential isoform abundance between known brain regions and analysis of spatial isoform patterns independent of predefined regions.

Main Results:

• Spl-ISO-Seq2 successfully captured isoform-level spatial variation in the adult mouse brain.
• Analysis revealed significant differential isoform abundance for genes like *Rps24* within specific cell types (e.g., oligodendrocytes).
• Spatial isoform patterns for genes such as *Snap25* were identified within excitatory neurons, and novel patterns for *Tnnc1* were discovered, highlighting cell-type-specific spatial expression.

Conclusions:

• The developed Spl-ISO-Seq2 technology and analytical pipeline provide unprecedented isoform-level resolution in spatial transcriptomics.
• These methods enable the discrimination of spatial variability within a single cell type from differences in cell-type abundance across regions.
• This work significantly advances the field by offering a detailed isoform view of spatial variation for individual cell types, crucial for understanding brain function and disease.