RT Journal Article SR Electronic T1 In Situ Transcriptome Accessibility Sequencing (INSTA-seq) JF bioRxiv FD Cold Spring Harbor Laboratory SP 722819 DO 10.1101/722819 A1 Daniel Fürth A1 Victor Hatini A1 Je H. Lee YR 2019 UL http://biorxiv.org/content/early/2019/08/05/722819.abstract AB Subcellular RNA localization regulates spatially polarized cellular processes, but unbiased investigation of its control in vivo remains challenging. Current hybridization-based methods cannot differentiate small regulatory variants, while in situ sequencing is limited by short reads. We solved these problems using a bidirectional sequencing chemistry to efficiently image transcript-specific barcode in situ, which are then extracted and assembled into longer reads using NGS. In the Drosophila retina, genes regulating eye development and cytoskeletal organization were enriched compared to methods using extracted RNA. We therefore named our method In Situ Transcriptome Accessibility sequencing (INSTA-seq). Sequencing reads terminated near 3’ UTR cis-motifs (e.g. Zip48C, stau), revealing RNA-protein interactions. Additionally, Act5C polyadenylation isoforms retaining zipcode motifs were selectively localized to the optical stalk, consistent with their biology. Our platform provides a powerful way to visualize any RNA variants or protein interactions in situ to study their regulation in animal development.