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| Status |
Public on Jul 26, 2012 |
| Title |
The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments |
| Organism |
Homo sapiens |
| Experiment type |
Other
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| Summary |
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. Additionally, 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 pre-treating cells with harringtonine to immobilize initiating ribosomes. The protocol we describe requires 5 - 7 days to generate a completed ribosome profiling sequencing library.
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| Overall design |
Ribosome profiling in cultured mammalian cells under three different footprinting conditions
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| Contributor(s) |
Ingolia NT, Brar GA, Rouskin S, McGeachy AM, Weissman JS |
| Citation(s) |
22836135 |
| Submission date |
May 03, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Nicholas T Ingolia |
| E-mail(s) |
nick@ingolia.org
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| Phone |
410 246 3025
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| Organization name |
Carnegie Institution
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| Department |
Embryology
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| Lab |
Ingolia
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| Street address |
3520 San Martin Drive
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21218 |
| Country |
USA |
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| Platforms (1) |
| GPL11154 |
Illumina HiSeq 2000 (Homo sapiens) |
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| Samples (3) |
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| Relations |
| BioProject |
PRJNA163111 |
| SRA |
SRP012648 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE37744_RAW.tar |
1.3 Mb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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