<p>Controlled gene expression programs have a crucial role in shaping cellular functions and activities. At the core of this process lies the RNA life cycle, ensuring protein products are synthesized in the right place at the right time. Here we detail an integrated protocol for imaging-based highly multiplexed in situ profiling of spatial transcriptome using antibody-based protein comapping (STARmap PLUS), spatial translatome mapping (RIBOmap) and spatiotemporal transcriptome mapping (TEMPOmap). These methods selectively convert targeted RNAs, ribosome-bound mRNAs or metabolically labeled RNAs to DNA amplicons with gene-unique barcodes, which are read out through in situ sequencing under a confocal microscope. Compared with other methods, they provide the analytical capacity to track the spatial and temporal dynamics of thousands of RNA species in intact cells and tissues. Our protocol can be readily performed in laboratories experienced in working with RNA and equipped with confocal microscopy instruments. The wet lab experiments in preparing the amplicon library take 2–3 d, followed by variable sequencing times depending on the sample size and target gene number. The spatially resolved single-cell profiles enable downstream analysis, including cell type classification, cell cycle identification and determination of RNA life cycle kinetic parameters through computational analysis guided by the established tutorials. This spatial omics toolkit will help users to better understand spatial and temporal RNA dynamics in heterogeneous cells and tissues.</p>