Profiling Nascent RNA Transcription with TT-Seq

Nascent RNA production is a dynamic activity in eukaryotic cells that faithfully reflects the transcriptional activities of a cell population at a specific time point. To capture and quantify dynamic transcriptional activity at high temporal resolution, several methods have been developed, including global or precision run-on sequencing (GRO/PRO-seq) and transient transcriptome sequencing (TT-seq). TT-seq uses in-cell metabolic labeling of RNAs with 4-thiouridine (4sU) to specifically enrich newly synthesized, nascent RNAs. This strategy enables real-time measurement of transcriptional dynamics, including initiation, elongation, and termination, across the entire genome. It also enables the sensitive detection of various noncoding, nascent RNAs produced from regulatory elements, such as promoters and enhancers. By combining short pulse labeling with high-throughput sequencing, TT-seq can provide insights into RNA synthesis/decay rates, enhancer activity, and the kinetics of transcriptional regulation in response to various stimuli. The method has become a valuable tool for studying transcriptional control and gene regulation. Here, we describe the entire TT-seq workflow. Our protocol was based on the original TT-seq protocol, but we made several changes that streamlined the workflow.