Rapid and Signal Crowdedness-Robust In-Situ Sequencing through Hybrid Block Coding

Technology development in spatial transcriptomics has propelled further understanding of cell types and their organization in tissues, opening new possibilities for researchers to explore transcript distributions at subcellular levels. However, current methods have limitations in either resolution, sensitivity, or speed. Here we demonstrate SPRINTseq (Spatially Resolved and signal-diluted Next-generation Targeted sequencing), a new in situ sequencing strategy that combines hybrid block coding and molecular dilution to resolve molecular crowdedness, overcoming a fundamental challenge that precluded simultaneous fast and sensitive high-resolution data acquisition. Our method recovered over 142 million transcripts in 453,843 cells from four whole mouse brain slices in less than two days. We then show how this high-resolution transcript map can be used to decipher cellular and subcellular molecular architecture of Alzheimer's disease in an unprecedented way. This improved spatial transcriptomics technology allowed us to link abnormal cellular behaviors and their organization in diseased tissue to changes in their subcellular mRNA distribution, generating new biological insights in health and disease. ### Competing Interest Statement The authors have declared no competing interest.