Nucleotide-resolution mapping of nascent RNA revisits the principles of transcriptional reorganization of the human genome upon signaling.

虽然人类信使核糖核酸的编目现在已经成为常规，但解剖新生的转录本一直是一个挑战。到目前为止，各种可用的方法通常都很费力，而且只关注转录组的一部分。我们最近推出了Factory RNA-seq，这是一种允许在低测序深度下全面捕获编码和非编码新生RNA的方法。然而，我们仍然缺乏对新生转录的时空部署及其与RNA加工的联系的了解。根据这一建议，我们的目标是进一步发展我们的工厂RNA-seq方法，以实现单核苷酸拆分和单细胞转录，并将其应用于两种细胞因子TNFpha和TGFbeta刺激前后的人原代内皮细胞。通过这种方式，我们将能够解决新生RNA生产中的拓扑和时间序列，评估它们参与转录活性位点的过程，并最终重新审视我们目前对哺乳动物转录组重组的理解，以前所未有的分辨率发出信号。

Although human mRNA cataloguing has now become routine, dissecting nascent transcriptomes has been a challenge. The various approaches available to date were, in general, laborious, and focused on just parts of the transcriptome. We recently introduced factory RNA-seq, a methodology that allows the comprehensive capturing of both coding and non-coding nascent RNA at low sequencing depth. However, we still lack understanding on the spatio-temporal deployment of nascent transcription and its connection to RNA processing. With this proposal we aim at further developing our factory RNA-seq approach to achieve single-nucleotide resolution and single-cell trascriptomes, and apply it to human primary endothelial cells before and after cytokine stimulation with two cytokines, TNFalpha and TGFbeta. This way we will be able to address topology and temporal succession in the production of nascent RNAs, assess their involvement in the active sites of transcription, and ultimately revisit our current understanding of mammalian transcriptome reorganization upon signaling at unprecedented resolution.