In Situ Transcriptome Accessibility Sequencing (INSTA-seq)

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 RNAprotein 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.

Spatially Resolved Transcriptomes—Next Generation Tools for Tissue Exploration

“…In 2019, the research group behind FISSEQ presented in situ Transcriptome Accessibility Sequencing (INSTA-Seq). [40] Here, sequencing in situ is used to map a regional barcode, while sequencing ex situ is done after extraction, to obtain full transcript information. Decoding the barcode is achieved via a bidirectional SBL chemistry, which sequence short reads (5 bp + 6 bp) from both ends of the cDNA fragment.…”

Uncovering an Organ’s Molecular Architecture at Single-Cell Resolution by Spatially Resolved Transcriptomics

“…FISSEQ detected over 8000 RNA species unbiasedly in cultured fibroblasts by reading 27 bases of each transcript. However, it failed to apply to tissue sections due to difficulties in generating long reads in situ [68]. Lately, Fürth and colleagues [68] proposed a novel approach, in situ transcriptome accessibility sequencing (INSTA-seq), allowing unbiased investigation of small regulatory variants that cannot be distinguished by current hybridization-based methods or in situ sequencing limited by short read.…”

“…The RCPs are sequenced by SOLiD sequencing with a 30-base read length (Figure I, bottom). Recently, the next-generation of FISSEQ termed INSTA-seq [68] has been launched. With longer reads assembled using NGS, the small regulatory variants that were previously unsolved due to short read limitations have been detected by FISSEQ 2 (INSTA-seq).…”

Unraveling Heterogeneity in Transcriptome and Its Regulation Through Single-Cell Multi-Omics Technologies

“…Another method known as APEX-seq/APEX-MS utilizes ascorbate peroxidase APEX2 to probe the spatial organization of the cellular transcriptome and proteome, respectively (Padrón et al, 2019). Furthermore, the road to spatial interactomes has been paved by in situ transcriptome accessibility sequencing (INSTA-seq), which allows mapping of RNA and RNA-protein interactions in situ (Furth et al, 2019).…”