Single-cell nascent RNA sequencing of homogeneous cell populations

Insights into transcriptional bursting kinetics and regulation have emerged from real-time nascent RNA imaging and analyses of static RNA counts over cells. However, transcriptome-wide inference of bursting parameters is still challenging, and it is debated whether nearby genes are prone to co-bursting. Here, we developed sensitive single-cell profiling of newly transcribed (or new) RNA in cells (NASC-seq2) that we applied to ten thousand individual primary mouse cells to shed new light on bursting dynamics and coordination. We demonstrate that inference from transient RNA counts improves the inference of synthesis rate (ksyn) and transcriptional off rate (koff), and we demonstrate that ksyn and not koff correlates with burst size. The inferred koff rates were magnitudes higher than the on rates across the whole transcriptome illustrating the universality of bursting within the mouse genome. Reports of transcriptional hubs and co-bursting have raised the question to what extent transcriptional bursting of proximal genes occurs fully independent. Analyses of the new RNA profiles at allelic resolution revealed that co-bursting does not appear more frequently than expected by chance, irrespective of chromosomal proximity. However, confounding correlations are frequently detected when disregarding the allelic resolution. Altogether, single-cell new RNA profiling critically improves the inference of transcriptional bursting parameters and demonstrates a lack of coordination of transcriptional bursting events in mammalian cells.