Modern methods of genome editing enable the rapid generation of mouse models to study the regulation of protein synthesis. At the same time, few options are available to study translation in rodents as the animal's complexity severely limits the repertoire of experimental tools. Here we describe a method to monitor translation in mice and other small animals. The technique is based on a ribosome profiling and specifically tailored toward measuring translation elongation. However, it can be easily applied for short upstream reading frames discovery. The advantage of this method is the ability to study translation in fully developed animals without extracting and subculturing cells, therefore, maintaining unperturbed physiological conditions.Robust mechanisms exist that serve to dynamically regulate the translation of mRNA into proteins across heterogeneous tissues. These processes ensure timely generation of proteins in quantities that scale with the demands of specific cell types. Importantly, this translational regulation occurs with spatiotemporal precision and is capable of recalibration as conditions change. Aberrant regulation of translation contributes to and exacerbates a wide range of diseases. Although dynamic control of translation is an essential and fundamental process shared by organisms, specific tissues and cell types can be differentially impacted by circumstances that challenge and impair basal translation, highlighting the heterogeneous nature of translational regulation. To understand how translation is differentially regulated during changing environments and across specific cells and tissues, methods capable of profiling translation in specific tissues and cells are crucial. Here, we describe a method for profiling genome-wide translation in specific tissues or cell types in Drosophila melanogaster, in which we combine ribosome affinity purification with ribosome profiling to enable a simplified protocol for robust analysis of translation in specific tissues.Protein synthesis is an essential process that affects major cellular functions including growth, energy production, cell signaling, and enzymatic reactions. However, how it is impacted by aging and how the translation of specific proteins is changed during the aging process remain understudied. Although yeast is a widely used model for studying eukaryotic aging, analysis of age-related translational changes using ribosome profiling in this organism has been challenging due to the need for isolating large quantities of old cells. Here, we provide a detailed protocol for genome-wide analysis of protein synthesis using ribosome profiling in replicatively aged yeast. By combining genetic enrichment of old cells with the biotin affinity purification step, this method allows large-scale isolation of aged cells sufficient for generating ribosome profiling libraries. We also describe a strategy for normalization of samples using a spike-in with worm lysates that permits quantitative comparison of absolute translation levels between young and old cells.Ribosome profiling is a genome-wide approach to map the positions of ribosomes on messenger RNAs. The abundance of ribosome-protected fragments can be used within condition to compare relative translation activities between different transcripts and between distinct conditions for the same transcript. A unified and routine method is currently lacking, however, to normalize between conditions for differences in global translation levels. Here we describe experimental and computational methods to use an orthogonal species spike-in, or internal standard, to enable absolute comparisons of translation activity between conditions. This simple modification of standard ribosome profiling provides a robust approach for accurately interpreting the effects of diverse genetic, chemical, and environmental perturbations of translation.Ribosome profiling, first developed in 2009, is the gold standard for quantifying and qualifying changes to translation genome-wide (Ingolia et al., Science, 2009). Though first designed and optimized in vegetative budding yeast, it has since been modified and specialized for use in diverse cellular states in yeast, as well as in bacteria, plants, human cells, and many other organisms (Ingolia et al. Science, 2009, reviewed in (Ingolia et al., Cold Spring Harb Perspect Biol, 2019; Brar and Weissman, Nat Rev Mol Cell Biol, 2015)). Here we report the current ribosome profiling protocol used in our lab to study genome-wide changes to translation in budding yeast undergoing the developmental process of meiosis (Brar et al., Science, 2012; Cheng et al., Cell, 2018). We describe this protocol in detail, including the following steps collection and flash freezing samples, cell lysis and extract preparation, sucrose gradient centrifugation and monosome collection, RNA extraction, library preparation, and library quality control. Almost every step presented here should be directly applicable to performing ribosome profiling in other eukaryotic cell types or cell states.Monitoring whole-genome translation and mRNA ribosome occupancy in vivo using ribosome profiling has proven to be a powerful tool for discovery of gene expression regulation, mechanisms of translation, and new open reading frames, in a wide range of different cell types in different organisms. Here we describe its application to the malaria parasite, Plasmodium falciparum. We present methods for intact polysome purification from parasite cultures, polysome digestion, monosome purification, ribosome footprint nucleic acid extraction, and Illumina library preparation.The knowledge of translation start sites is crucial for annotation of genes in bacterial genomes. However, systematic mapping of start codons in bacterial genes has mainly relied on predictions based on protein conservation and mRNA sequence features which, although useful, are not always accurate. We recently found that the pleuromutilin antibiotic retapamulin (RET) is a specific inhibitor of translation initiation that traps ribosomes specifically at start codons, and we used it in combination with ribosome profiling to map start codons in the Escherichia coli genome. This genome-wide strategy, that was named Ribo-RET, not only verifies the position of start codons in already annotated genes but also enables identification of previously unannotated open reading frames and reveals the presence of internal start sites within genes. Here, we provide a detailed Ribo-RET protocol for E.  https://www.selleckchem.com/products/daratumumab.html  coli. Ribo-RET can be adapted for mapping the start codons of the protein-coding sequences in a variety of bacterial species.