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

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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.