Qi, H.; Anant, M. M.-G.; Faltine-Gonzalez, D. Z.; Hu, R.; Wei, L.; Workman, C. D.; Shi, C.; Del Rosario, I.; Kebschull, J. M.

Understanding cellular identity requires multimodal measurements in single cells. Cellular barcoding provides powerful tools for recording the properties or history of individual cells in nucleic acids, while spatial omics techniques enable the measurement of a growing list of molecular features at micron resolution in tissue. However, existing methods that integrate these approaches in single samples are limited in the modalities they support, their flexibility, and efficiency. Here, we present BARseq3, a modular system that combines cellular barcoding with high-efficiency spatial transcriptomics and translatomics at subcellular resolution in tissue. BARseq3 is compatible with fixed samples, immunostaining, diverse species, and can be easily extended to include other spatial assays, enabling a multimodal understanding of cellular identity.