To improve our understanding of pattern formation during development and disease we heavily rely on the identification of novel regulators and pathways. While RNA sequencing yields genome-wide expression data that suit this purpose, it lacks spatial resolution. Such spatial resolution can be obtained by microscopy-based methods like in situ hybridization, but these fail to provide information on more than a few genes at a time. Here, we describe tomo-seq, a technique that combines the advantages of the above-mentioned approaches and provides genome-wide expression data with spatial information. The tomo-seq technique is based on cryosectioning of an embryo or tissue of interest and performing RNA-seq on individual sections. Using this method, we have generated genome-wide transcriptomics with high spatial resolution of the whole zebrafish embryo at various stages of development (Junker et al., 2014) and of adult zebrafish hearts after injury (Wu et al., 2016).