High-Resolution Profiling of NMD Targets in Yeast

Contemporary high-throughput sequencing methods, notably RNA-Seq, permit the systematic identification and characterization of transcripts whose levels change significantly in response to altered biological states. We have described methods for the application of this methodology to a definition of the transcripts regulated by the NMD pathway in the yeast Saccharomyces cerevisiae. In short, we outline methods for growing cells of wild-type or NMD-deficient yeast, isolating RNA from the different strains, depleting rRNA from each sample, preparing and sequencing the respective RNA-Seq libraries, and employing multiple software packages to characterize the resulting sequence reads meaningfully. Our experimental approach has identified approximately 900 transcripts that are commonly upregulated when yeast NMD is inactivated (Celik, Baker, He, & Jacobson, 2017).