Single-cell RNA sequencing offers snapshots of whole transcriptomes but obscures the temporal dynamics of RNA biogenesis and decay. Here we present single-cell metabolically labeled new RNA tagging sequencing (scNT-Seq), a method for massively parallel analysis of newly-transcribed and pre-existing RNAs from the same cell. This droplet microfluidics-based method enables high-throughput chemical conversion on barcoded beads, efficiently marking newly-transcribed RNAs with T-to-C substitutions. The steps of the protocol are (1) metabolically labeling of cells with 4sU, (2) co-encapsulating individual cell with a barcoded oligo-dT primer coated bead in a nanoliter-scale droplet, (3) performing one-pot 4sU chemical conversion on pooled barcoded beads, and (4) reverse transcription, cDNA amplification, tagmentation, indexing PCR, and sequencing. scNT-Seq provides a broadly applicable strategy to investigate dynamic biological systems at single-cell resolution.