Profiling of HeLa nucleoplasmic and nucleolar RNAs by Halo-seq proximity labeling

Abstract Subcellular RNA localization is a key regulator of gene expression, but transcriptome wide characterization of RNAs enriched in specific cellular compartments has been hindered by methodological limitations in throughput and spatial resolution. Halo-seq is an RNA proximity-labeling approach that enables the extraction of RNAs located near virtually any Halo-tagged bait protein. However, its broader application has been restricted by the limited availability of the required Halo ligand. Here we present a Halo-seq dataset profiling the nucleoplasmic and nucleolar transcriptomes of HeLa cells. Through experimental and computational validation, we demonstrate that our optimized Halo-seq protocol achieves high spatial specificity, enabling robust mapping of compartment-enriched RNA populations.