Genome‐Wide Annotation and Quantitation of Translation by Ribosome Profiling

Abstract Protein translation represents a key regulated step in gene expression. Here, we present procedures for comprehensive and quantitative analysis of translation in vivo. This approach, termed ribosome profiling, makes it possible to directly measure the rate of protein synthesis and to reveal the full range of proteins translated by the cell, including unexpected micropeptides and alternative protein isoforms. Ribosome profiling relies on nuclease footprinting of ribosomes onto mRNAs followed by the analysis of these ribosome‐protected fragments by deep sequencing. The protocol presented here encompasses technical optimizations to capture ribosomes at their positions in vivo, perform high‐resolution nuclease footprinting, and prepare deep‐sequencing libraries from these footprints. The alignment of ribosome footprint sequences is discussed as well. The presence of aligned ribosome footprints indicates ribosome occupancy and thus translation, and the density of these footprints reports on the rate of translation. The unit also discusses the use of the translation inhibitor harringtonine to immobilize initiating ribosomes specifically and thereby map sites of translation initiation. Experimental manipulations for ribosome profiling take roughly 5 to 7 days, and sequencing and analysis require an additional 4 to 5 days. © 2013 by John Wiley & Sons, Inc.