ABSTRACT
Single-cell RNA sequencing offers snapshots of whole transcriptomes but obscures the temporal dynamics of RNA biogenesis and decay. Here we present single-cell nascent transcript tagging sequencing (scNT-Seq), a method for massively parallel analysis of nascent and pre-existing RNAs from the same cell. This droplet microfluidics-based method enables high-throughput chemical conversion on barcoded beads, efficiently marking metabolically labeled nascent transcripts with T-to-C substitutions. By simultaneously measuring nascent and pre-existing transcriptomes, scNT-Seq reveals neuronal subtype-specific gene regulatory networks and time-resolved RNA trajectories in response to brief (minutes) versus sustained (hours) neuronal activation. Integrating scNT-Seq with genetic perturbation reveals that DNA methylcytosine dioxygenases may inhibit stepwise transition from pluripotent embryonic stem cell state to intermediate and totipotent two-cell-embryo-like (2C-like) states by promoting global nascent transcription. Furthermore, pulse-chase scNT-Seq enables transcriptome-wide measurements of RNA stability in rare 2C-like cells. Time-resolved single-cell transcriptomic analysis thus opens new lines of inquiry regarding cell-type-specific RNA regulatory mechanisms.