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Biotechniques. 2015 Jun 1;58(6):329-32. doi: 10.2144/000114302. eCollection 2015 Jun.

A streamlined ribosome profiling protocol for the characterization of microorganisms.

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Bioengineering Department, University of California, San Diego, La Jolla, CA.
Fuels Synthesis Division, Joint BioEnergy Institute, Emeryville, CA.
Department of Chemical & Biomolecular Engineering, Department of Bioengineering, University of California, Berkeley, Berkeley, CA.
QB3 Institute, University of California, Emeryville, CA.
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark.


Ribosome profiling is a powerful tool for characterizing in vivo protein translation at the genome scale, with multiple applications ranging from detailed molecular mechanisms to systems-level predictive modeling. Though highly effective, this intricate technique has yet to become widely used in the microbial research community. Here we present a streamlined ribosome profiling protocol with reduced barriers to entry for microbial characterization studies. Our approach provides simplified alternatives during harvest, lysis, and recovery of monosomes and also eliminates several time-consuming steps, in particular size-selection steps during library construction. Furthermore, the abundance of rRNAs and tRNAs in the final library is drastically reduced. Our streamlined workflow enables greater throughput, cuts the time from harvest to the final library in half (down to 3-4 days), and generates a high fraction of informative reads, all while retaining the high quality standards of the existing protocol.


Illumina; bacteria; next-generation sequencing; ribosome profiling

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