In Situ Transcriptome Accessibility Sequencing (INSTA-seq)

Citations Over Time

Abstract

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.

Related Papers

• → Next-Generation Sequencing for MicroRNA Expression Profile(2017)77 cited
• → High Throughput Sequencing for the Detection and Characterization of RNA Viruses(2021)72 cited
• → Ultra-deep mutant spectrum profiling: improving sequencing accuracy using overlapping read pairs(2013)49 cited
• → Emulsion PCR-coupled target enrichment: An effective fishing method for high-throughput sequencing of poorly preserved ancient DNA(2013)20 cited
• → Analysis of Quasispecies of Avain Leukosis Virus Subgroup J Using Sanger and High-throughput Sequencing(2016)4 cited