Ribosome profiling has emerged as a technique for measuring translation comprehensively and quantitatively by deep sequencing of ribosome-protected mRNA fragments. By identifying the precise positions of ribosomes, footprinting experiments have unveiled key insights into the composition and regulation of the expressed proteome, including delineating potentially functional micropeptides, revealing pervasive translation on cytosolic RNAs, and identifying differences in elongation rates driven by codon usage or other factors. This Primer looks at important experimental and analytical concerns for executing ribosome profiling experiments and surveys recent examples where the approach was developed to explore protein biogenesis and homeostasis.
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