title: The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments.
abstract: Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. We present a protocol for genome-wide, quantitative analysis of in vivo translation by deep sequencing. This ribosome profiling approach maps the exact positions of ribosomes on transcripts by nuclease footprinting. The nuclease-protected mRNA fragments are converted into a DNA library suitable for deep sequencing using a strategy that minimizes bias. The abundance of different footprint fragments in deep sequencing data reports on the amount of translation of a gene. In addition, footprints reveal the exact regions of the transcriptome that are translated. To better define translated reading frames, we describe an adaptation that reveals the sites of translation initiation by pretreating cells with harringtonine to immobilize initiating ribosomes. The protocol we describe requires 5-7 days to generate a completed ribosome profiling sequencing library. Sequencing and data analysis require a further 4-5 days.
authors: Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California, USA. ingolia@ciwemb.edu
authors: Nicholas T
authors: NT
authors: Ingolia
authors: Gloria A
authors: GA
authors: Brar
authors: Silvia
authors: S
authors: Rouskin
authors: Anna M
authors: AM
authors: McGeachy
authors: Jonathan S
authors: JS
authors: Weissman
mesh_terms: D000818:Animals; D001483:Base Sequence; D015723:Gene Library; D006248:Harringtonines / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D014176:Protein Biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012335:RNA, Ribosomal; D012260:Ribonucleases / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology; D017423:Sequence Analysis, RNA / Q000379:methods*; D059467:Transcriptome
chemical_list: D006248:Harringtonines; D012333:RNA, Messenger; D012335:RNA, Ribosomal; C062500:harringtonin; D012260:Ribonucleases