title: Transcriptome-wide measurement of ribosomal occupancy by ribosome profiling.
abstract: 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.
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland.
authors.forename: Florian
authors.initials: F
authors.lastname: Aeschimann
authors.affiliation: Max Planck Institute for Biology of Ageing, Joseph-Stelzmann-Straße 9b, 50931 Cologne, Germany.
authors.forename: Jieyi
authors.initials: J
authors.lastname: Xiong
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Arnold
authors.affiliation: Max Planck Institute for Biology of Ageing, Joseph-Stelzmann-Straße 9b, 50931 Cologne, Germany. Electronic address: christoph.dieterich@age.mpg.de.
authors.forename: Christoph
authors.initials: C
authors.lastname: Dieterich
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland. Electronic address: helge.grosshans@fmi.ch.
authors.forename: Helge
authors.initials: H
authors.lastname: Großhans
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D012270:Ribosomes / Q000235:genetics*; D059467:Transcriptome / Q000235:genetics*
chemical_list: D029742:Caenorhabditis elegans Proteins
keywords: Caenorhabditis elegans; Polysome profiling; Post-transcriptional regulation; Ribosome profiling; Translation; Translational control