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 new transcript 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 metabolically labeled newly-transcribed RNAs with T-to-C substitutions. By simultaneously measuring new and old 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 RNA biogenesis. 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.
Footnotes
We have made minor changes to the terminology in the updated manuscript. Since nascent transcripts are conventionally defined as RNAs that are be synthesized by RNA polymerase II before it is cleaved at the polyA cleavage site, we decided to replace "nascent" with "new/newly-transcribed" to more accurately describe the metabolically labeled mRNAs captured by our method.
