TY - JOUR T1 - Massively parallel, time-resolved single-cell RNA sequencing with scNT-Seq JF - bioRxiv DO - 10.1101/2019.12.19.882050 SP - 2019.12.19.882050 AU - Qi Qiu AU - Peng Hu AU - Kiya W. Govek AU - Pablo G. Camara AU - Hao Wu Y1 - 2019/01/01 UR - http://biorxiv.org/content/early/2019/12/20/2019.12.19.882050.abstract N2 - 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. ER -