In Situ Transcriptome Accessibility Sequencing (INSTA-seq)

Fürth, Daniel, Hatini, Victor, Lee, Je (August 2019) In Situ Transcriptome Accessibility Sequencing (INSTA-seq). BioRxiv. (Unpublished)

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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.

Item Type:	Paper
Subjects:	Investigative techniques and equipment > INSTA-seq
CSHL Authors:	Lee, Je Hyuk Furth, Daniel
Communities:	CSHL labs > Lee lab
SWORD Depositor:	CSHL Elements
Depositing User:	CSHL Elements
Date:	5 August 2019
Date Deposited:	07 May 2021 20:44
Last Modified:	29 Apr 2024 15:27
URI:	https://repository.cshl.edu/id/eprint/40069

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