Ribosome Profiling in the Model Diatom Thalassiosira pseudonana

Pichler, Monica ORCID: https://orcid.org/0000-0002-5162-2534, Meindl, Andreas, Romberger, Markus, Eckes-Shephard, Annemarie, Nyberg-Brodda, Carl Fredrik, Buhigas, Claudia, Llaneza-Lago, Sergio, Lehmann, Gerhard, Hopes, Amanda, Meister, Gunter, Medenbach, Jan and Mock, Thomas ORCID: https://orcid.org/0000-0001-9604-0362 (2023) Ribosome Profiling in the Model Diatom Thalassiosira pseudonana. Current Protocols, 3 (7). ISSN 2691-1299

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Abstract

Diatoms are an important group of eukaryotic microalgae, which play key roles in marine biochemical cycling and possess significant biotechnological potential. Despite the importance of diatoms, their regulatory mechanisms of protein synthesis at the translational level remain largely unexplored. Here, we describe the detailed development of a ribosome profiling protocol to study translation in the model diatom Thalassiosira pseudonana, which can easily be adopted for other diatom species. To isolate and sequence ribosome-protected mRNA, total RNA was digested, and the ribosome-protected fragments were obtained by a combination of sucrose-cushion ultracentrifugation and polyacrylamide gel electrophoresis for size selection. To minimize rRNA contamination, a subtractive hybridization step using biotinylated oligos was employed. Subsequently, fragments were converted into sequencing libraries, enabling the global quantification and analysis of changes in protein synthesis in diatoms. The development of this novel ribosome profiling protocol represents a major expansion of the molecular toolbox available for diatoms and therefore has the potential to advance our understanding of the translational regulation in this important group of phytoplankton.

Item Type:	Article
Additional Information:	Funding Information: This work was supported by the UKRI‐BBSRC Norwich Research Park Biosciences Doctoral Training Partnership (grant BB/M011216/1 to MP) and the German Research Foundation (grant SFB960 TP B11 to JM). We thank EMBL GeneCore for excellent support with sequencing. Funding Information: This work was supported by the UKRI-BBSRC Norwich Research Park Biosciences Doctoral Training Partnership (grant BB/M011216/1 to MP) and the German Research Foundation (grant SFB960 TP B11 to JM). We thank EMBL GeneCore for excellent support with sequencing. Publisher Copyright: © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.
Uncontrolled Keywords:	diatoms,high-throughput sequencing,ribosome profiling,rna,thalassiosira pseudonana,translation,neuroscience(all),biochemistry, genetics and molecular biology(all),immunology and microbiology(all),pharmacology, toxicology and pharmaceutics(all),health informatics,medical laboratory technology,sdg 14 - life below water ,/dk/atira/pure/subjectarea/asjc/2800
Faculty \ School:	Faculty of Science > School of Environmental Sciences Faculty of Medicine and Health Sciences > Norwich Medical School University of East Anglia Research Groups/Centres > Theme - ClimateUEA
UEA Research Groups:	Faculty of Science > Research Centres > Centre for Ecology, Evolution and Conservation Faculty of Science > Research Groups > Centre for Ocean and Atmospheric Sciences Faculty of Science > Research Groups > Environmental Biology
Related URLs:	http://www.scopus.com/inward/record.url?...
Depositing User:	LivePure Connector
Date Deposited:	11 Mar 2024 10:31
Last Modified:	11 Mar 2024 10:31
URI:	https://ueaeprints.uea.ac.uk/id/eprint/94620
DOI:	10.1002/cpz1.843

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