Aeschimann F Xiong J Arnold A Dieterich C Großhans H 2015 Gene expression profiling provides a tool to analyze the internal states of cells or organisms, and their responses to perturbations. While global measurements of mRNA levels have thus been widely used for many years, it is only through the recent development of the ribosome profiling technique that an analogous examination of global mRNA translation programs has become possible. Ribosome profiling reveals which RNAs are being translated to what extent and where the translated open reading frames are located. In addition, different modes of translation regulation can be distinguished and characterized. Here, we present an optimized, step-by-step protocol for ribosome profiling. Although established in Caenorhabditis elegans, our protocol and optimization approaches should be equally usable for other model organisms or cell culture with little adaptation. Next to providing a protocol, we compare two different methods for isolation of single ribosomes and two different library preparations, and describe strategies to optimize the RNase digest and to reduce ribosomal RNA contamination in the libraries. Moreover, we discuss bioinformatic strategies to evaluate the quality of the data and explain how the data can be analyzed for different applications. In sum, this article seeks to facilitate the understanding, execution, and optimization of ribosome profiling experiments. https://sonar.ch/global/documents/84070 eng info:eu-repo/semantics/altIdentifier/doi/10.1016/j.ymeth.2015.06.013 info:eu-repo/semantics/altIdentifier/pmid/26102273 Methods (San Diego, Calif.). - 2015 Caenorhabditis elegans Polysome profiling Post-transcriptional regulation Ribosome profiling Translation Translational control Animals Caenorhabditis elegans Caenorhabditis elegans Proteins Gene Expression Profiling Ribosomes Transcriptome Transcriptome-wide measurement of ribosomal occupancy by ribosome profiling. http://purl.org/coar/resource_type/c_6501