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| Status |
Public on May 12, 2009 |
| Title |
Affinity purification of ribosomes and associated RNAs using tagged Rpl16a and Rpl16b |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Other
|
| Summary |
In this study, we systematically identified RNAs associated with ribosomes. To identify ribosome associated RNAs, C-terminal ZZ-tagged Rpl16a or Rpl16b, expressed under control of thier native promoter, were affinity purified from whole cell extracts of cultures grown to mid-log phase in minimal medium. Extracts were incubated with immunoglobulin G (IgG) coupled microbeads, washed, and ribosomes were eluted by tobacco etch virus (TEV) protease treatment. We performed two biological replicates with each protein and analyzed the RNA content using oligo microarrays. Total RNA isolated from extracts of cells expressing Rpl16a-ZZ or Rpl16b-ZZ (input) and from the affinity-purified ribosomes was reverse transcribed with oligo(dT) primers. cDNA was labeled with Cy3 and Cy5 fluorescent dyes, respectively, and competitively hybridized to yeast oligo microarrays. Alternatively, RNA was isolated from sucrose gradient fractions containing 60S subunits, 80S monosomes and polysomes. Here, we performed four biological replicates. Analysis was done with oligo microarrays as described above.
A strain or line experiment design type assays differences between multiple strains, cultivars, serovars, isolates, lines from organisms of a single species.
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| |
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| Overall design |
strain_or_line_design
|
| |
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| Contributor(s) |
Halbeisen R |
| Citation(s) |
19419242 |
| |
| Submission date |
Nov 18, 2008 |
| Last update date |
Mar 20, 2012 |
| Contact name |
Andre Gerber |
| E-mail(s) |
andre.gerber@pharma.ethz.ch
|
| Phone |
+41 44 6337378
|
| Fax |
+41 44 6337364
|
| Organization name |
Swiss Federal Institute of Technology
|
| Department |
Institute of Pharmaceutical Science
|
| Street address |
Wolfgang Pauli Str. 10, HCI H396
|
| City |
ETH Hoenggerberg, Zuerich |
| ZIP/Postal code |
ZH |
| Country |
Switzerland |
| |
|
| Platforms (1) |
| GPL8546 |
SMD Print_1548 Saccharomyces cerevisiae |
|
| Samples (8)
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| This SubSeries is part of SuperSeries: |
| GSE13682 |
Affinity purification of ribosomes and associated RNAs from stress-treated cells using tagged Rpl16a and Rpl16b |
|
| Relations |
| BioProject |
PRJNA113917 |
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