Single-cell nascent RNA sequencing using click-chemistry unveils coordinated transcription
Transcription is the primary regulatory step in gene expression coordinating the coding and non-coding genomic regions across space and time. Divergent initiation of transcription from promoters and enhancers produces stable RNAs from genes and unstable RNAs from enhancers. Nascent RNA capture and sequencing assays simultaneously measure gene and enhancer activity from cell populations. However, the lack of single-cell resolved view of active transcription has left fundamental questions in gene regulation unanswered. In this study, we present scGRO-seq - a single-cell nascent RNA sequencing assay using copper-catalyzed azide-alkyne cycloaddition - unveiling the coordinated regulation of dynamic transcription throughout the genome. scGRO-seq demonstrates the episodic nature of transcription and provides estimates of burst size and frequency by directly quantifying transcribing RNA polymerases. It reveals the co-transcription of functionally related genes and leverages the transcription of replication-dependent non-polyadenylated histone genes to elucidate cell-cycle dynamics. The single-nucleotide spatiotemporal resolution of scGRO-seq characterizes networks of enhancers and genes and indicates the bursting of transcription at super-enhancers before the activation of burst from associated genes. Our method offers insights into the dynamic nature of transcription, functional architecture of the genome, and serves as a powerful tool to investigate the role of the non-coding genome in gene regulation. Overall design: Nascent RNA in mouse embryonic stem cells were labelled with propargyl (click-chemistry compatible moiety) via nuclear run-on reaction. Individual intact nuclei after labelling were placed in 96-well plate and nascent RNA was conjugated to single-stranded DNA containing single-cell barcode by copper-catalyzed azide-alkyne cycloaddition. Single-cell barcoded nascent RNA from wells of a 96-well plate were pooled and NGS library was prepared and sequenced.