Preparation of RNA 3' End Sequencing Libraries of Total and 4-thiouracil Labeled RNA for Simultaneous Measurement of Transcription, RNA Synthesis and Decay in S. cerevisiae.

Cellular RNA levels are determined by the rates of RNA transcription from the gene template and subsequent RNA stability. Knowledge about both transcription and RNA decay is, therefore, necessary to interpret RNA levels and gene expression, especially during cellular processes where these parameters change. Numerous experimental strategies have been developed to measure transcription and RNA decay rates. However, to our knowledge, none of those techniques can simultaneously interrogate transcription and RNA decay. The presented protocol allows this and provides a simple approach to simultaneously estimate total RNA levels, transcription and decay rates from the same RNA sample. It is based on brief metabolic labeling of RNA and subsequent concurrent sequencing of polyA+ and polyA- RNA 3' ends. The protocol was developed in S. cerevisiae and should be broadly applicable.