Optimized protocol for quantifying 5′ UTR-mediated translation initiation in S. cerevisiae using direct analysis of ribosome targeting
Summary: 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, purific...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-12-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166722007420 |
| _version_ | 1811199389094379520 |
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| author | Cole J.T. Lewis Rachel O. Niederer Ritam Neupane Wendy V. Gilbert |
| author_facet | Cole J.T. Lewis Rachel O. Niederer Ritam Neupane Wendy V. Gilbert |
| author_sort | Cole J.T. Lewis |
| collection | DOAJ |
| description | Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-04-12T01:47:31Z |
| format | Article |
| id | doaj.art-af981bb5ca9f45f6927277ea97495b77 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-04-12T01:47:31Z |
| publishDate | 2022-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-af981bb5ca9f45f6927277ea97495b772022-12-22T03:53:02ZengElsevierSTAR Protocols2666-16672022-12-0134101862Optimized protocol for quantifying 5′ UTR-mediated translation initiation in S. cerevisiae using direct analysis of ribosome targetingCole J.T. Lewis0Rachel O. Niederer1Ritam Neupane2Wendy V. Gilbert3Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, New Haven, CT 06520, USADepartment of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USADepartment of Molecular Biophysics and Biochemistry, Yale School of Medicine, New Haven, CT 06520, USADepartment of Molecular Biophysics and Biochemistry, Yale School of Medicine, New Haven, CT 06520, USA; Corresponding authorSummary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166722007420SequencingMolecular BiologyGene ExpressionSystems biology |
| spellingShingle | Cole J.T. Lewis Rachel O. Niederer Ritam Neupane Wendy V. Gilbert Optimized protocol for quantifying 5′ UTR-mediated translation initiation in S. cerevisiae using direct analysis of ribosome targeting STAR Protocols Sequencing Molecular Biology Gene Expression Systems biology |
| 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 | Sequencing Molecular Biology Gene Expression Systems biology |
| url | http://www.sciencedirect.com/science/article/pii/S2666166722007420 |
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