|
| Status |
Public on Jan 23, 2017 |
| Title |
Synechocystis_PCC6803_∆ssaA_4h_N_recovery_1 |
| Sample type |
RNA |
| |
|
| Source name |
∆ssaA_4h
|
| Organism |
Synechocystis sp. PCC 6803 |
| Characteristics |
genotype: {delta}ssaA
protocol: nitrogen recovery
time: 4h
|
| Treatment protocol |
For nitrogen depletion, NaNO3 was omitted from the medium and cells were first washed twice and then resuspended in nitrogen-free medium. Cultures were incubated in nitrogen-depleted medium for 7 days. To induce nitrogen recovery, starved cells were supplemented with 17.6 mM NaNO3. Cells were harvested after cultivation in nitrogen-repleted medium for the indicated times (1h, 4h, 22h, 48h, 144h), to document recovery.
|
| Growth protocol |
The Synechocystis sp PCC 6803 strain PCC-M (Trautmann, 2012) used in this study was provided by S. Shestakov (Moscow State University) and cultivated on 1% (w/v) agar (Bacto agar; Difco) plates containing BG-11 mineral medium (Rippka, 1979). Liquid cultures of wild-type and mutant strains were grown in BG-11 medium containing 10 mM TES buffer, pH 8.0, under continuous illumination with white light at 80 mmol photons m–2 s–1 at 30 °C, supplemented with a continuous stream of ambient air. BG11 plates for the cultivation of mutant strains were supplemented with 40 µg ml-1 kanamycin (∆ssaA). No antibiotics were added to the liquid cultures to eliminate any influence.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For the isolation of RNA, samples from discrete stages of cultivation were taken at the indicated times and were immediately put on ice and spun down at 4 °C. The preparation of total RNA from Synechocystis 6803 was essentially performed as described previously (Dienst et al., 2008), using the Hot Trizol method, followed by 2-propanol precipitation.
|
| Label |
Cy3
|
| Label protocol |
The DNAse treated RNA 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.
|
| |
|
| Hybridization protocol |
The labelled RNA was fragmented and hybridized as described by the manufacturer's instructions for Agilent one color microarrays with 1.65 µg of labeled RNA
|
| Scan protocol |
Arrays were scanned on the Agilent Technologies Scanner G2505C US90900275, using Agilent Feature Extraction Software 10.7.3.1 and the protocols GE1_107_Sep09 for Cy3 labelled arrays
|
| Data processing |
Raw data were processed with the R package Limma. Median signal intensity was normexp background corrected and quantile normalized.
|
| |
|
| Submission date |
Jan 23, 2017 |
| Last update date |
Jan 23, 2017 |
| Contact name |
Jens Georg |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Freiburg
|
| Street address |
Schänzlestr. 1
|
| City |
Freiburg |
| ZIP/Postal code |
79104 |
| Country |
Germany |
| |
|
| Platform ID |
GPL15867 |
| Series (1) |
| GSE83387 |
6S RNA plays a role in recovery from nitrogen depletion in Synechocystis sp. PCC 6803 |
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