Spatially resolved single-cell translatomics at molecular resolution.

The precise control of messenger RNA (mRNA) translation is a crucial step in posttranscriptional gene regulation of cellular physiology. However, it remains a challenge to systematically study mRNA translation at the transcriptomic scale with spatial and single-cell resolution. Here, we report the development of ribosome-bound mRNA mapping (RIBOmap), a highly multiplexed three-dimensional in situ profiling method to detect cellular translatome. RIBOmap profiling of 981 genes in HeLa cells revealed cell cycle-dependent translational control and colocalized translation of functional gene modules. We mapped 5413 genes in mouse brain tissues, yielding spatially resolved single-cell translatomic profiles for 119,173 cells and revealing cell type-specific and brain region-specific translational regulation, including translation remodeling during oligodendrocyte maturation. Our method detected widespread patterns of localized translation in neuronal and glial cells in intact brain tissue networks.