The evolution of insects has been marked by the appearance of key body plan innovations that promoted the outstanding ability of this lineage to adapt to new habitats, boosting the most successful radiation in animals. To understand the evolution of these new structures, it is essential to investigate which are the genes and gene regulatory networks participating during the embryonic development of insects. Great efforts have been made to fully understand gene expression and gene regulation during the development of holometabolous insects, in particular Drosophila melanogaster. Conversely, functional genomics resources and databases in other insect lineages are scarce. To provide a new platform to study gene regulation in insects, we generated ATAC-seq (Assay for transposase-Accessible Chromatin) for the first time during the development of the mayfly Cloeon dipterum, which belongs to Paleoptera, the sister group to all other winged insects. With these comprehensive datasets along six developmental stages, we characterised pronounced changes in accessible chromatin between early and late embryogenesis. The application of ATAC-seq in mayflies provides a fundamental resource to understand the evolution of gene regulation in insects.

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