Ribosome Profiling in the Model Diatom Thalassiosira pseudonana
(2023) In Current protocols 3(7).- 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... (More)
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.
(Less)
- author
- organization
- publishing date
- 2023-07-01
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- diatoms, high-throughput sequencing, ribosome profiling, RNA, Thalassiosira pseudonana, translation
- in
- Current protocols
- volume
- 3
- issue
- 7
- article number
- e843
- publisher
- John Wiley & Sons Inc.
- external identifiers
-
- pmid:37439534
- scopus:85164540656
- ISSN
- 2691-1299
- DOI
- 10.1002/cpz1.843
- language
- English
- LU publication?
- yes
- additional info
- Publisher Copyright: © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.
- id
- 3d404ff7-1808-4c4c-b02d-73b59213537d
- date added to LUP
- 2023-07-26 11:00:41
- date last changed
- 2024-10-05 17:03:40
@article{3d404ff7-1808-4c4c-b02d-73b59213537d, abstract = {{<p>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.</p>}}, author = {{Pichler, Monica and Meindl, Andreas and Romberger, Markus and Eckes-Shephard, Annemarie and Nyberg-Brodda, Carl Fredrik and Buhigas, Claudia and Llaneza-Lago, Sergio and Lehmann, Gerhard and Hopes, Amanda and Meister, Gunter and Medenbach, Jan and Mock, Thomas}}, issn = {{2691-1299}}, keywords = {{diatoms; high-throughput sequencing; ribosome profiling; RNA; Thalassiosira pseudonana; translation}}, language = {{eng}}, month = {{07}}, number = {{7}}, publisher = {{John Wiley & Sons Inc.}}, series = {{Current protocols}}, title = {{Ribosome Profiling in the Model Diatom Thalassiosira pseudonana}}, url = {{http://dx.doi.org/10.1002/cpz1.843}}, doi = {{10.1002/cpz1.843}}, volume = {{3}}, year = {{2023}}, }