UC Irvine

The subcellular localization of specific RNA molecules promotes localized cellular activity across a variety of species and cell types. The misregulation of this RNA targeting can result in developmental defects, and mutations in proteins that regulate this process are associated with multiple diseases. For the vast majority of localized RNAs, however, the mechanisms that underlie their subcellular targeting are unknown, partly due to the difficulty associated with profiling and quantifying subcellular RNA populations. To address this challenge, we developed Halo-seq, a proximity labeling technique that can label and profile local RNA content at virtually any subcellular location. Halo-seq relies on a HaloTag fusion protein localized to a subcellular space of interest. Through the use of a radical-producing Halo ligand, RNAs that are near the HaloTag fusion are specifically labeled with spatial and temporal control. Labeled RNA is then specifically biotinylated in vitro via a click reaction, facilitating its purification from a bulk RNA sample using streptavidin beads. The content of the biotinylated RNA is then profiled using high-throughput sequencing. In this article, we describe the experimental and computational procedures for Halo-seq, including important benchmark and quality control steps. By allowing the flexible profiling of a variety of subcellular RNA populations, we envision Halo-seq facilitating the discovery and further study of RNA localization regulatory mechanisms. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Visualization of HaloTag fusion protein localization Basic Protocol 2: In situ copper-catalyzed cycloaddition of fluorophore via click reaction Basic Protocol 3: In vivo RNA alkynylation and extraction of total RNA Basic Protocol 4: In vitro copper-catalyzed cycloaddition of biotin via click reaction Basic Protocol 5: Assessment of RNA biotinylation by RNA dot blot Basic Protocol 6: Enrichment of biotinylated RNA using streptavidin beads and preparation of RNA-seq library Basic Protocol 7: Computational analysis of Halo-seq data.