Global Run-On sequencing to measure nascent transcription in Saccharomyces cerevisiae

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.

Keywords: Nascent RNA; RNA polymerase; Sequencing; Transcription Run-On; Transcription directionality; Transcription initiation; Transcription termination.

Figure 1:. Experimental set up of GRO-seq protocol in budding yeast.

The detailed workflow of the GRO-seq method outlining the major steps of the protocol.

Figure 2:. Quantitative and qualitative analysis of cDNA library preparations.

(A) Automated gel electrophoresis profile depicting size distribution of the cDNA library. Lane A1 is the ladder while lane A2 is cDNA library lane. (B) Quantification of ladder in lane A1 in Figure 2A. (C) Size distribution of cDNA library A2 from Figure 2A showing size range and peak of cDNA library. Lower and upper markers from the ladder are included. The region between the vertical blue lines is set on either side of the library to calculate the size of the library within this region and is used for normalization prior to sequencing.

Figure 3:. Sample of GRO-seq data view on the University of California Santa Cruz (UCSC) genome browser showing reads aligned to a region of chromosome 1 harboring three genes CYS3, SWC3 and MDM10.

The figure was generated using a bigWig track depicting the read coverage. The aligned reads were converted to genome coverage using bedtools genomecov and then converted to bigwig format using USCS’s bigWig to bedGraph program before displaying in the UCSC genome browser.