title: Simple and inexpensive ribosome profiling analysis of mRNA translation.
abstract: The development and application of ribosome profiling has markedly advanced our understanding of ribosomes and mRNA translation. The experimental approach, which relies on deep sequencing of ribosome-protected mRNA fragments generated by treatment of polyribosomes with exogenous nucleases, provides a transcriptome-wide assessment of translation. The broad application of ribosome profiling has been slowed by the complexity and expense of the protocol. Here, we provide a simplified ribosome profiling method that uses micrococcal nuclease to generate ribosome footprints in crude cellular extracts, which are then purified simply by size selection via polyacrylamide gel electrophoresis. This simplification removes the laborious or expensive purification of ribosomes that has typically been used. This direct extraction method generates gene-level ribosome profiling data that are similar to a method that includes ribosome purification. This protocol should significantly ease the barrier to entry for research groups interested in employing ribosome profiling.
authors.affiliation: Programme in Cardiovascular and Metabolic Disorders, Duke-NUS Graduate Medical School, Singapore 169857, Singapore. Electronic address: david.reid@duke-nus.edu.sg.
authors.forename: David W
authors.initials: DW
authors.lastname: Reid
authors.affiliation: Programme in Cardiovascular and Metabolic Disorders, Duke-NUS Graduate Medical School, Singapore 169857, Singapore; Programme in Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School, Singapore 169857, Singapore.
authors.forename: Shirish
authors.initials: S
authors.lastname: Shenolikar
authors.affiliation: Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: Christopher V
authors.initials: CV
authors.lastname: Nicchitta
mesh_terms: D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism; D008836:Micrococcal Nuclease; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D012333:RNA, Messenger; D008836:Micrococcal Nuclease
keywords: Deep sequencing; Ribosome profiling; Transcriptomics; mRNA