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Updated: Aug 15, 2025

Quantitative Immunofluorescence to Measure Global Localized Translation
Published on: August 22, 2017
Optimized protocol for quantifying 5' UTR-mediated translation initiation in S. cerevisiae using direct analysis of
Cole J T Lewis1, Rachel O Niederer2, Ritam Neupane1
1Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, New Haven, CT 06520, USA.
Abstract:
Direct analysis of ribosome targeting (DART) allows investigators to measure the translation initiation potential of thousands of RNAs in parallel. Here, we describe an optimized protocol for generating active translation extract from S. cerevisiae, followed by in vitro translation, purification of ribosome-bound RNAs, and subsequent library preparation and sequencing. This protocol can be applied to a variety of cell types and will enable high-throughput interrogation of translational determinants. For complete details on the use and execution of this protocol, please refer to Niederer et al. (2022).1.
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