Application of high-throughput 5′P sequencing for the study of co-translational mRNA decay

Summary mRNA degradation is connected to the translation process up to the degree that 5′-3′ mRNA degradation follows the last translating ribosome. To study 5′-3′co-translational mRNA decay and the associated ribosome dynamics, here we present an improved high-throughput 5′P degradome RNA sequencing protocol (HT-5Pseq). We exemplify its application in Saccharomyces cerevisiae, but in principle, it could be applied to any other eukaryotic organism. HT-5Pseq is easy, scalable, and uses affordable duplex-specific nuclease-based rRNA depletion. For complete details on the use and execution of this protocol, please refer to Zhang and Pelechano (2021) .