Department of Cell and Molecular Biology, Karolinska Instiutet, Biomedicum, Solnavägen 9, 171 65, Solna, Sweden.
Department of Biosciences and Nutrition, Karolinska Institutet, NEO, Blickagången 16, 141 52, Huddinge, Sweden.
Nat Commun. 2019 Jul 17;10(1):3138. doi: 10.1038/s41467-019-11028-9.
Sequencing of newly synthesised RNA can monitor transcriptional dynamics with great sensitivity and high temporal resolution, but is currently restricted to populations of cells. Here, we develop new transcriptome alkylation-dependent single-cell RNA sequencing (NASC-seq), to monitor newly synthesised and pre-existing RNA simultaneously in single cells. We validate the method on pre-labelled RNA, and by demonstrating that more newly synthesised RNA was detected for genes with known high mRNA turnover. Monitoring RNA synthesis during Jurkat T-cell activation with NASC-seq reveals both rapidly up- and down-regulated genes, and that induced genes are almost exclusively detected as newly transcribed. Moreover, the newly synthesised and pre-existing transcriptomes after T-cell activation are distinct, confirming that NASC-seq simultaneously measures gene expression corresponding to two time points in single cells. Altogether, NASC-seq enables precise temporal monitoring of RNA synthesis at single-cell resolution during homoeostasis, perturbation responses and cellular differentiation.
新合成 RNA 的测序可以非常灵敏和高时间分辨率地监测转录动态，但目前仅限于细胞群体。在这里，我们开发了新的转录组依赖于烷基化的单细胞 RNA 测序（NASC-seq），以在单细胞中同时监测新合成和预先存在的 RNA。我们通过对预先标记的 RNA 进行验证，并通过证明对于已知高 mRNA 周转率的基因，检测到更多新合成的 RNA，验证了该方法的有效性。使用 NASC-seq 在 Jurkat T 细胞激活期间监测 RNA 合成，揭示了快速上调和下调的基因，并且诱导基因几乎仅被检测为新转录。此外，T 细胞激活后新合成和预先存在的转录组是不同的，这证实了 NASC-seq 可以同时在单细胞中测量对应于两个时间点的基因表达。总之，NASC-seq 能够在稳态、扰动反应和细胞分化过程中以单细胞分辨率精确地监测 RNA 合成的时间进程。
