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In eukaryotic cells, RNA biogenesis generally requires processing of the nascent transcript as it is being synthesized by RNA polymerase. These processing events include endonucleolytic cleavage, exonucleolytic trimming, and splicing of the growing nascent transcript. Endonucleolytic cleavage events that generate an exposed 5′-monophosphorylated (5′-PO4) end on the growing nascent transcript occur in the maturation of rRNAs, tRNAs, and mRNAs. These 5′-PO4 ends can be a target of further processing or be subjected to 5′-3′ exonucleolytic digestion that may result in termination of transcription. Here, we describe how to identify 5′-PO4 ends of intermediates in nascent RNA metabolism. We capture these species via metabolic labeling with bromouridine followed by immunoprecipitation and specific ligation of 5′-PO4 RNA ends with the 3′-hydroxyl group of a 5′ adaptor (5′-PO4 Bru-Seq) using RNA ligase I. These ligation events are localized at single nucleotide resolution via highthroughput sequencing, which identifies the position of 5′-PO4 groups precisely. This protocol successfully detects the 5′monophosphorylated ends of RNA processing intermediates during production of mature ribosomal, transfer, and micro RNAs. When combined with inhibition of the nuclear 5′-3′ exonuclease Xrn2, 5′-PO4 Bru-Seq maps the 5′ splice sites of debranched introns and mRNA and tRNA 3′ end processing sites cleaved by CPSF73 and RNaseZ, respectively.Key features• Metabolic labeling for brief periods with bromouridine focuses the analysis of 5′-PO4 RNA ends on the population of nascent transcripts that are actively transcribed.• Detects 5′-PO4 RNA ends on nascent transcripts produced by all RNA polymerases.• Detects 5′-PO4 RNA ends at single nucleotide resolution.