Battistoni, G.; Garcia, S. T.; Sia, C. Y.; Corriero, S.; Boquetale, C.; Williams, E. G.; Alini, M.; Hemmer, N.; IMAXT Cancer Grand Challenge Consortium, ; Balasubramanian, S.; Nicholson, B. C.; Hannon, G. J.; Bressan, D.

Mapping the molecular identities and functions of cells within their spatial context is key to understanding the complex interplay within and between tissue neighbourhoods. A wide range of methods have recently enabled spatial profiling of cellular anatomical contexts, some offering single-cell resolution. These use different barcoding schemes to encode either the location or the identity of target molecules. However, all these technologies face a trade-off between spatial resolution, depth of profiling, and scalability. Here, we present Barcoding by Activated Linkage of Indexes (BALI), a method that uses light to write combinatorial spatial molecular barcodes directly onto target molecules in situ, enabling multi-omic profiling by next generation sequencing. A unique feature of BALI is that the user can define the number, size, and shape, and resolution of the spatial locations to be interrogated, with the potential to profile millions of distinct regions with subcellular precision. As a proof of concept, we used BALI to capture the transcriptome, chromatin accessibility, or both simultaneously, from distinct areas of the mouse brain in single tissue sections, demonstrating strong concordance with publicly available datasets. BALI therefore combines high spatial resolution, high throughput, histological compatibility, and workflow accessibility to enable powerful spatial multi-omic profiling