Optimized protocol for quantifying 5′ UTR-mediated translation initiation in S. cerevisiae using direct analysis of ribosome targeting

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...

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Detalles Bibliográficos
Autores principales: Lewis, Cole J.T., Niederer, Rachel O., Neupane, Ritam, Gilbert, Wendy V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9678775/
https://www.ncbi.nlm.nih.gov/pubmed/36595943
http://dx.doi.org/10.1016/j.xpro.2022.101862
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author Lewis, Cole J.T.
Niederer, Rachel O.
Neupane, Ritam
Gilbert, Wendy V.
author_facet Lewis, Cole J.T.
Niederer, Rachel O.
Neupane, Ritam
Gilbert, Wendy V.
author_sort Lewis, Cole J.T.
collection PubMed
description 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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spelling pubmed-96787752022-11-23 Optimized protocol for quantifying 5′ UTR-mediated translation initiation in S. cerevisiae using direct analysis of ribosome targeting Lewis, Cole J.T. Niederer, Rachel O. Neupane, Ritam Gilbert, Wendy V. STAR Protoc Protocol 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) Elsevier 2022-11-17 /pmc/articles/PMC9678775/ /pubmed/36595943 http://dx.doi.org/10.1016/j.xpro.2022.101862 Text en © 2022 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Lewis, Cole J.T.
Niederer, Rachel O.
Neupane, Ritam
Gilbert, Wendy V.
Optimized protocol for quantifying 5′ UTR-mediated translation initiation in S. cerevisiae using direct analysis of ribosome targeting
title Optimized protocol for quantifying 5′ UTR-mediated translation initiation in S. cerevisiae using direct analysis of ribosome targeting
title_full Optimized protocol for quantifying 5′ UTR-mediated translation initiation in S. cerevisiae using direct analysis of ribosome targeting
title_fullStr Optimized protocol for quantifying 5′ UTR-mediated translation initiation in S. cerevisiae using direct analysis of ribosome targeting
title_full_unstemmed Optimized protocol for quantifying 5′ UTR-mediated translation initiation in S. cerevisiae using direct analysis of ribosome targeting
title_short Optimized protocol for quantifying 5′ UTR-mediated translation initiation in S. cerevisiae using direct analysis of ribosome targeting
title_sort optimized protocol for quantifying 5′ utr-mediated translation initiation in s. cerevisiae using direct analysis of ribosome targeting
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9678775/
https://www.ncbi.nlm.nih.gov/pubmed/36595943
http://dx.doi.org/10.1016/j.xpro.2022.101862
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