Single-cell RNA sequencing offers snapshots of whole transcriptomes but obscures the temporal RNA dynamics. 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 mRNAs from the same cell. This droplet microfluidics-based method enables high-throughput chemical conversion on barcoded beads, efficiently marking newly transcribed mRNAs with T-to-C substitutions. Using scNT-seq, we jointly profiled new and old transcriptomes in ~55,000 single cells. These data revealed time-resolved transcription factor activities and cell-state trajectories at the single-cell level in response to neuronal activation. We further determined rates of RNA biogenesis and decay to uncover RNA regulatory strategies during stepwise conversion between pluripotent and rare totipotent two-cell embryo (2C)-like stem cell states. Finally, integrating scNT-seq with genetic perturbation identifies DNA methylcytosine dioxygenase as an epigenetic barrier into the 2C-like cell state. Time-resolved single-cell transcriptomic analysis thus opens new lines of inquiry regarding cell-type-specific RNA regulatory mechanisms.

单细胞 RNA 测序提供了整个转录组的快照，但掩盖了时间 RNA 动态。在这里，我们介绍了单细胞代谢标记的新 RNA 标记测序 (scNT-seq)，这是一种对来自同一细胞的新转录和预先存在的 mRNA 进行大规模并行分析的方法。这种基于液滴微流体的方法能够在条形码珠子上进行高通量化学转化，有效地标记具有 T 到 C 替换的新转录的 mRNA。使用 scNT-seq，我们在约 55,000 个单细胞中联合分析了新旧转录组。这些数据揭示了响应神经元激活的单细胞水平的时间分辨转录因子活性和细胞状态轨迹。我们进一步确定了 RNA 生物发生和衰变的速率，以揭示多能和罕见的全能双细胞胚胎 (2C) 样干细胞状态之间逐步转换过程中的 RNA 调节策略。最后，将 scNT-seq 与遗传扰动相结合，将 DNA 甲基胞嘧啶双加氧酶鉴定为进入 2C 样细胞状态的表观遗传屏障。因此，时间分辨单细胞转录组学分析开辟了有关细胞类型特异性 RNA 调节机制的新研究路线。