The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments

• Published in: Nature protocols Vol. 7; no. 8; pp. 1534 - 1550
• Main Authors: Ingolia, Nicholas T, Brar, Gloria A, Rouskin, Silvia, McGeachy, Anna M, Weissman, Jonathan S
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.08.2012; Nature Publishing Group
• Subjects: 38/39; 45/91; 631/1647/2217/2018; 631/1647/514/1949; 631/337/574/1789; Analytical Chemistry; Animals; Base Sequence; Biological Techniques; Computational Biology/Bioinformatics; Data analysis; Deoxyribonucleic acid; DNA; DNA sequencing; Footprinting; Fragments; Gene expression; Gene Library; Gene mapping; Genetic translation; Genomes; Harringtonines - pharmacology; Humans; In vivo methods and tests; Life Sciences; Measurement techniques; Messenger RNA; Microarrays; Molecular Sequence Data; Nuclease; Organic Chemistry; Peptide Chain Initiation, Translational; Peptides; Physiological aspects; Protein Biosynthesis - genetics; Proteins; Protocol; Reading; Ribonucleases - metabolism; Ribonucleic acid; Ribosomes; Ribosomes - drug effects; Ribosomes - genetics; Ribosomes - metabolism; RNA; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Ribosomal; Saccharomyces cerevisiae - cytology; Sequence Analysis, RNA - methods; Transcriptome; Transcriptomes; Translation; Translation initiation; United States; San Francisco California; California; United States > US; Maryland; Baltimore Maryland
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2012.086

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.