Abstract
Global Run-On sequencing is a reliable and widely used approach for monitoring nascent transcription on a genomewide scale. The assay has been successfully used for studying global transcription in humans, plants, worms, flies, and fission yeast. Here we describe a GRO-seq protocol for studying transcription in budding yeast, Saccharomyces cerevisiae. Briefly, the technique involves permeabilization of actively growing yeast cells, allowing transcription to proceed in permeabilized cells in the presence of brominated UTP, affinity purification of bromo-UMP incorporated nascent transcripts followed by cDNA library construction, deep sequencing, and mapping against the reference genome. The approach maps the position of transcriptionally active RNA polymerase on a genomewide basis. In addition to identifying the complete set of transcriptionally active genes in a cell under a given set of conditions, the method can be used to determine elongation rate, termination defect and promoter directionality at the genomewide level. The approach is especially useful in identifying short-lived unstable transcripts that are rapidly degraded even before they leave the nucleus.