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A simple, fast, and cost-efficient protocol for ultra-sensitive ribosome profiling

Ribosome profiling has become an essential tool for studying messenger RNA (mRNA) translation in cells with codon-level resolution. However, its widespread application remains hindered by the labour-intensive workflow, low efficiency, and high costs associated with sequencing sample preparation. Her...

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Main Authors: Koubek, Jiří (Author) , Jetzinger, Katharina (Author) , Dror, Shiran (Author) , Irastortza-Olaziregi, Mikel (Author) , Frank, Dana (Author) , Kotan, Ilgin (Author) , Santos, Jaime (Author) , Tippmann, Frank (Author) , Lafrenz, Pascal (Author) , Kaessmann, Henrik (Author) , Amster-Choder, Orna (Author) , Bukau, Bernd (Author) , Kramer, Günter (Author)
Format: Article (Journal)
Language:English
Published: 17 September 2025
In: Nucleic acids research
Year: 2025, Volume: 53, Issue: 17, Pages: 1-12
ISSN:1362-4962
DOI:10.1093/nar/gkaf902
Online Access:Verlag, kostenfrei, Volltext: https://doi.org/10.1093/nar/gkaf902
Verlag, kostenfrei, Volltext: https://academic.oup.com/nar/article/53/17/gkaf902/8256616?login=true
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Author Notes:Jiří Koubek, Katharina Jetzinger, Shiran Dror, Mikel Irastortza-Olaziregi, Dana Frank, Ilgin Kotan, Jaime Santos, Frank Tippmann, Pascal Lafrenz, Henrik Kaessmann, Orna Amster-Choder, Bernd Bukau, Günter Kramer
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Summary:Ribosome profiling has become an essential tool for studying messenger RNA (mRNA) translation in cells with codon-level resolution. However, its widespread application remains hindered by the labour-intensive workflow, low efficiency, and high costs associated with sequencing sample preparation. Here, we present a new cost-effective and ultra-sensitive library preparation method that significantly advances the applicability of ribosome profiling. By implementing bead-coupled enzymatic reactions and product purifications, our approach increases both yield and throughput while maintaining high reproducibility. Demonstrating the sensitivity of the protocol, we prepared libraries from as little as 12 fmol of RNA, which expands the feasibility of ribosome profiling from minimal input samples, such as those derived from small populations, stressed cells, or patient-derived specimens. Additionally, we validate the versatility of the protocol across multiple species and demonstrate its applicability for RNA-seq library preparation. Altogether, this protocol provides a highly accessible and efficient alternative to existing ribosome profiling workflows, facilitating research in previously challenging experimental contexts.
Item Description:Gesehen am 03.02.2026
Physical Description:Online Resource
ISSN:1362-4962
DOI:10.1093/nar/gkaf902

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