Skip to main content

Poly-A Tailing and Adaptor Ligation Methods for Ribo-Seq Library Construction

Part of the Methods in Molecular Biology book series (MIMB,volume 2252)

Abstract

Ribosome profiling is a powerful technique that enables researchers to monitor translational events across the transcriptome. It provides a snapshot of ribosome positions and density across the transcriptome at a sub-codon resolution. Here we describe the whole procedure of profiling ribosome footprints in mammalian cells. Two methods for Ribo-seq library construction are introduced, and their advantages and disadvantages are compared. There is a room for further improvement of Ribo-seq in terms of the amount of starting material, the duration of library construction, and the resolution of sequencing results.

Key words

  • Ribo-seq
  • Translation
  • mRNA
  • Codon
  • Deep sequencing

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-0716-1150-0_10
  • Chapter length: 17 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   119.00
Price excludes VAT (USA)
  • ISBN: 978-1-0716-1150-0
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   159.99
Price excludes VAT (USA)
Hardcover Book
USD   219.99
Price excludes VAT (USA)
Fig. 1
Fig. 2

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Ingolia NT, Ghaemmaghami S, Newman JR, Weissman JS (2009) Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324(5924):218–223. https://doi.org/10.1126/science.1168978

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  2. Brar GA, Weissman JS (2015) Ribosome profiling reveals the what, when, where and how of protein synthesis. Nat Rev Mol Cell Biol 16(11):651–664. https://doi.org/10.1038/nrm4069

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  3. Wolin SL, Walter P (1988) Ribosome pausing and stacking during translation of a eukaryotic mRNA. EMBO J 7(11):3559–3569

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  4. Stern-Ginossar N, Weisburd B, Michalski A, Le VT, Hein MY, Huang SX, Ma M, Shen B, Qian SB, Hengel H, Mann M, Ingolia NT, Weissman JS (2012) Decoding human cytomegalovirus. Science 338(6110):1088–1093. https://doi.org/10.1126/science.1227919

    CAS  CrossRef  PubMed  Google Scholar 

  5. Ingolia NT, Lareau LF, Weissman JS (2011) Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147(4):789–802. https://doi.org/10.1016/j.cell.2011.10.002

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  6. Gao X, Wan J, Liu B, Ma M, Shen B, Qian SB (2015) Quantitative profiling of initiating ribosomes in vivo. Nat Methods 12(2):147–153. https://doi.org/10.1038/nmeth.3208

    CAS  CrossRef  PubMed  Google Scholar 

  7. Gerashchenko MV, Gladyshev VN (2014) Translation inhibitors cause abnormalities in ribosome profiling experiments. Nucleic Acids Res 42(17):e134. https://doi.org/10.1093/nar/gku671

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  8. Santos DA, Shi L, Tu BP, Weissman JS (2019) Cycloheximide can distort measurements of mRNA levels and translation efficiency. Nucleic Acids Res 47(10):4974–4985. https://doi.org/10.1093/nar/gkz205

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  9. Hornstein N, Torres D, Das Sharma S, Tang G, Canoll P, Sims PA (2016) Ligation-free ribosome profiling of cell type-specific translation in the brain. Genome Biol 17(1):149. https://doi.org/10.1186/s13059-016-1005-1

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  10. Turchinovich A, Surowy H, Serva A, Zapatka M, Lichter P, Burwinkel B (2014) Capture and Amplification by Tailing and Switching (CATS). An ultrasensitive ligation-independent method for generation of DNA libraries for deep sequencing from picogram amounts of DNA and RNA. RNA Biol 11(7):817–828. https://doi.org/10.4161/rna.29304

    CrossRef  PubMed  PubMed Central  Google Scholar 

  11. Ingolia NT, Brar GA, Rouskin S, McGeachy AM, Weissman JS (2012) The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat Protoc 7(8):1534–1550. https://doi.org/10.1038/nprot.2012.086

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  12. McGlincy NJ, Ingolia NT (2017) Transcriptome-wide measurement of translation by ribosome profiling. Methods 126:112–129. https://doi.org/10.1016/j.ymeth.2017.05.028

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  13. Reid DW, Shenolikar S, Nicchitta CV (2015) Simple and inexpensive ribosome profiling analysis of mRNA translation. Methods 91:69–74. https://doi.org/10.1016/j.ymeth.2015.07.003

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported by the US National Institutes of Health (R01GM1222814 and R21CA227917) and HHMI Faculty Scholar (55108556).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Shu-Bing Qian .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Verify currency and authenticity via CrossMark

Cite this protocol

Qiu, J., Qian, SB. (2021). Poly-A Tailing and Adaptor Ligation Methods for Ribo-Seq Library Construction. In: Labunskyy, V.M. (eds) Ribosome Profiling. Methods in Molecular Biology, vol 2252. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1150-0_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1150-0_10

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1149-4

  • Online ISBN: 978-1-0716-1150-0

  • eBook Packages: Springer Protocols