Abstract
Conventional protocols for inferring gene regulatory networks from spatial transcriptomics data often neglect spatial constraints. Here, we present a protocol using SpaGRN, a statistical framework integrating extracellular signaling and spatial dependencies, to reconstruct spatially aware receptor-transcription factor-target regulatory cascades. This protocol details steps for data preprocessing, spatial autocorrelation, co-expression construction, cis-regulatory motif enrichment, receptor-associated regulon inference, and 3D visualization. This approach enables the systematic mapping of signaling pathways in complex tissues such as developing embryos and tumors. For complete details on the use and execution of this protocol, please refer to Li et al.(1).