Abstract
Transcription occurring at gene loci results in accumulation of mature RNA molecules (i.e., mRNAs) that can be easily assayed by RT-PCR or RNA sequencing. However, the steady-state level of RNA does not accurately mirror transcriptional activity per se. In fact, RNA stability plays a major role in determining the relative abundance of any given RNA molecule. Here, I describe a protocol of Nuclear Run-On assay coupled to deep sequencing to assess real-time transcription from engaged RNA polymerase. Mapping nascent transcripts at the genome-wide scale provides a reliable measure of transcriptional activity in mammalian cells and delivers a high-resolution map of coding and noncoding transcripts that is especially useful for annotation and quantification of short-lived RNA molecules.