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.
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Change history
17 August 2012
In the version of this article initially published, the table in Step 32 of the protocol lists “T4 PNK (10 U µl–1)”, “1.0 µl” and “10 U” in the last row. This entry should read “T4 Rnl2(tr) (200 U µl–1)”, “1.0 µl” and “200 U”. The error has been corrected in the HTML and PDF versions of the article.
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Acknowledgements
We thank members of the Weissman and Ingolia labs, as well as H. Guo, D. Bartel, S. Luo and G. Schroth for advice in developing this protocol. This work was supported by the US National Institutes of Health (NIH) through an NIH P01 grant (AG10770; to J.S.W.) and a Ruth L. Kirschstein National Research Service Award (GM080853; to N.T.I.), an American Cancer Society postdoctoral fellowship (117945-PF-09-136-01-RMC; to G.A.B.) and the Searle Scholars Program (N.T.I.)
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N.T.I. and J.S.W. designed the study. G.A.B. and J.S.W. developed the rRNA depletion protocol. S.R. and J.S.W. adapted the protocol to use preadenylylated linker ligation. N.T.I., S.R., G.A.B. and A.M.M. performed experiments. N.T.I. and A.M.M. analyzed the data. N.T.I. and J.S.W. wrote the manuscript.
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N.T.I. and J.S.W. are inventors on a patent covering the technique described in this manuscript.
Supplementary information
Supplementary Fig. 1
Comparison of expression measurements in different buffer conditions. The lower left-hand triangle in the comparison matrix compares per-gene expression measurements under two different buffer conditions, as in Fig. 2b. The upper right-hand triangle shows the histogram of log2 ratios in the comparison, as in Fig. 2b. (PDF 4635 kb)
Supplementary Fig. 2
Reading frame information in HEK293 samples prepared with different buffer conditions. (a-c) Stacked histograms showing the fraction of footprint reads at each length, separated based on the reading frame position of the 5′ end of the read, relative to the first codon nucleotide. (d-f) Histogram of footprint reads at each length, and of the information content of footprints at that length. The information content is defined as the difference between the entropy of the position distribution with no reading frame information, in which any of three codon positions are equally likely, and the entropy of the position distribution with reading frame information. (PDF 135 kb)
Supplementary Note
Galaxy workflow for ribosome footprinting analysis. This file contains a workflow that demonstrates the preprocessing and alignment of one million sequencing reads taken from the data presented here. This workflow requires the Galaxy software48. (ZIP 105251 kb)
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Ingolia, N., Brar, G., Rouskin, S. et al. The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat Protoc 7, 1534–1550 (2012). https://doi.org/10.1038/nprot.2012.086
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DOI: https://doi.org/10.1038/nprot.2012.086
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