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Massively parallel and time-resolved RNA sequencing in single cells with scNT-seq.

Nature methods (2020-09-02)
Qi Qiu, Peng Hu, Xiaojie Qiu, Kiya W Govek, Pablo G Cámara, Hao Wu
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
聚-L-鸟氨酸 盐酸盐, mol wt 15,000-30,000
Sigma-Aldrich
尿核甙, BioUltra, ≥99%