|
| Status |
Public on Jun 18, 2014 |
| Title |
Mutant RNA Sample 17.5_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Mutant RNA Sample 17.5
|
| Organism |
Synechocystis sp. PCC 6803 |
| Characteristics |
strain: PCC 6803
timepoint: 17.5
genotype: deletion of kaiAB1C1
|
| Growth protocol |
Cells were grown in liquid cultures under constant illumination with white light at 50 µmol photons m^-2 s^-1 in BG11 medium (Rippka et al., 1979) supplemented with 10 mM TES pH 8 at 30 °C. After three days, cultures were shifted to light-dark conditions (12h:12h light:dark cycle). Cells were harvested by filtering through Supor-800 0.8 µm membrane filters during the first period of alternated illumination at timepoints CT 17.5 (timepoint 17.5) and CT 5.5 (timepoint 5.5) and after six days of growth in a light-dark cycle at timepoint CT 5.5 (timepoint 0).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Each membrane filter was immediately put into PGTX reagent (Pinto et al., 2009), fragmented by vortexing, frozen in liquid nitrogen and stored at ―20 °C. The samples were thawed at 95 °C and vortexed in between. After incubation on ice for 5 min, 150 µl of cold bromochloropropane were added. Following an incubation at room temperature for 15 min, the phases were separated by centrifugation at 4 °C. The aqueous phase was treated with an equal volume of phenol/chloroform/isoamylalcohol (25:24:1) and centrifuged at 4°C. The RNA in the supernatant was precipitated over night at ―20 °C by addition of an equal volume of isopropanol, pelleted by centrifugation at 13.000 rpm for 30 min at 4°C, washed with 1 ml of 75 % v/v ethanol and pelleted again. The RNA was air dried and resuspended in DEPC-treated ddH2O. The total extracted RNA was quantified using a Nanodrop spectrophotometer. Prior to the microarray analysis, the RNA was treated with DNaseI (Invitrogen). Integrity of the RNA was examined by electrophoretic separation on a 1.3% (w/v) formaldehyde agarose gel.
|
| Label |
Cy3
|
| Label protocol |
The RNA mix was labeled directly, without cDNA synthesis in 2 µg aliquots with the Kreatech “ULS labeling kit for Agilent gene expression arrays” with Cy3 according to the manufacturers protocol. DNA was fragmented by 3h incubation at 95°C in H2O and Cy3 labeled with the Kreatech kit mentioned before.
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| |
|
| Hybridization protocol |
For RNA hybridizations, the labelled RNA mix was fragmented and hybridized as described by the manufacturer's instructions for Agilent one color microarrays with 1.65 µg of labeled RNA. Hybridization of labelled genomic DNA was performed similar to RNA hybridization, but omittingt the fragmentation step in the Agilent protocol. For DNA hybridization 0.5 to 3.8 µg of labeled DNA were used.
|
| Scan protocol |
Arrays were scanned on the Agilent Technologies Scanner G2505B, using Agilent Feature Extraction Software 10.7.3.1 and the modified protocol GE1_107_Sep09 Cy3 labelled arrays
|
| Data processing |
Raw data were processed with the R package Limma. Median signal intensity was quantile normalized.
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| |
|
| Submission date |
Jun 17, 2014 |
| Last update date |
Jun 18, 2014 |
| Contact name |
Jan Mitschke |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Freiburg
|
| Department |
Biologie III
|
| Lab |
AG Wilde
|
| Street address |
Schaenzlestr. 1
|
| City |
Freiburg |
| ZIP/Postal code |
79104 |
| Country |
Germany |
| |
|
| Platform ID |
GPL15867 |
| Series (1) |
| GSE58572 |
Deletion of the Synechocystis sp. PCC 6803 kaiAB1C1 gene cluster causes impaired cell growth under light-dark conditions |
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