LAUSR

Peroxidase‐generated proximity labeling is in widespread use to study subcellular proteomes and the protein interaction networks in living cells, but the development of subcellular RNA labeling is limited. APEX‐seq has emerged as a new method to study subcellular RNA in living cells, but the labeling of RNA still has room to improve. In this work, we describe 4‐thiouridine (s4U)‐enhanced peroxidase‐generated biotinylation of RNA with high efficiency. The incorporation of s4U could introduce additional sites for RNA labeling, enhanced biotinylation was observed on monomer, model oligo RNA and total RNA. Through the s4U metabolic approach, the in vivo RNA biotinylation efficiency by peroxidase catalysis was also dramatically increased, which will benefit RNA isolation and study for the spatial transcriptome.