Whole-brain Spatial Validation of Single-cell Gene Expression with Subcellular Resolution

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Various scRNA-seq approaches enable comprehensive screening of complete tissues but require tissue dissociation and the removal of individual cells from their biological context. Validation of differential gene expression in situ is, therefore, of high interest. Rebus Biosystems has developed an automated platform that combines Synthetic Aperture Optics, fluidics engineering, and single-molecule RNA fluorescence in-situ hybridization (smRNA FISH) chemistry to probe up to 30 gene targets across a large tissue sections with subcellular resolution. Here we demonstrate the capabilities of the platform in a mouse brain tissue section. smRNA FISH probes were designed to detect each gene of interest and multiplexed sequentially on the tissue section. Imaging data was collected, processed, and analyzed to quantify individual RNA molecules across 100,000+ single cells in an ~0.5 cm2 tissue section. Our imaging data accurately reproduced cell type abundance as determined from brain atlas data and validated the expected spatial location of different cell types within the whole tissue. These data exhibit a powerful and robust automated platform that can translate biological insights from transcriptomic screening experiments into a spatial context. ​