Protocol for high-resolution 3D spatial transcriptomics using Open-ST

Spatial transcriptomics (ST) is fundamental for understanding molecular mechanisms in health and disease. Here, we present a protocol for efficient and high-resolution ST in 2D/3D with Open-ST. We describe all steps for repurposing Illumina flow cells into spatially barcoded capture areas and preparing ST libraries from stained cryosections. We detail the computational workflow for generating 2D/3D molecular maps ("virtual tissue blocks"), aligned with histological data, unlocking molecular pathways in space. Open-ST is applicable to any tissue, including clinical samples. For complete details on the use and execution of this protocol, please refer to Schott et al.¹.