|
| Status |
Public on Jun 15, 2016 |
| Title |
PCC6803_WT_14d_N-depletion_1 |
| Sample type |
RNA |
| |
|
| Source name |
14d_N_depletion
|
| Organism |
Synechocystis sp. PCC 6803 |
| Characteristics |
genotype: WT
|
| Treatment protocol |
Nitrogen starvation was induced by washing exponentially growing cells once with BG11 medium lacking sodium nitrate (BG110) and transferring them into BG110 at an OD750 of 0.4 as described previously (Schlebusch and Forchhammer, 2010). The recovery was triggered by pelleting the cells and transferring the starved cells into BG11 at an OD750 of 0.4.
|
| Growth protocol |
Synechocystis strains used in this study were propagated in BG11 medium supplemented with 5 mM NaHCO3, as described previously (Rippka et al., 1979). In liquid culture all strains were grown under continuous illumination with white light in a range of 40 to 50 μmol photons m−2 s−1 at 27°C. The growth of the culture was monitored via the optical density at 750 nm wavelength (OD750). Nitrogen starvation was induced by washing exponentially growing cells once with BG11 medium lacking sodium nitrate (BG110) and transferring them into BG110 at an OD750 of 0.4 as described previously (Schlebusch and Forchhammer, 2010). The recovery was triggered by pelleting the cells and transferring the starved cells into BG11 at an OD750 of 0.4.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA for tailing arrays was extracted via the PGTX method adapted from Pinto et al (Pinto et al., 2009). Therefore, 15 ml cyanobacterial culture (OD750 ~1.0) was rapidly harvested on Supor®-800 membranes (Pall; Life Sciences; 0.8µm, 47mm) with a vacuum pump assembly. The folded membrane was then transferred in 2 ml screw lid vial containing ~250 µl sterile glass beads and 1 ml PGTX, vortexed shortly, snap frozen in liquid nitrogen and stored at -80°C until continuing with the extraction. The samples were homogenized in a “FastPrep®-24” (MP Biomedicals) 4 times for 15 s at 4°C and incubated at 65°C for at least 15 min while shaking. For 1 ml of PGTX mixture 0,.6 ml of a Chloroformchloroform:Isoamyl isoamyl alcohol mixture (24:1) was added and mixed for 10 min before centrifugation at 4000 x g for 15 min. The supernatant was transferred into sterile RNase-free 2 ml cups and mixed with an equal amount ofand the c Chloroform:Isoamyl isoamyl alcohol mixture (24:1) before another centrifugation step for 15 min at 4000 xgextraction repeated. 0.3 µl of RNA grade glycogen as well as anthe equal amount volume of isopropanol was added to the supernatant before incubation at -80°C overnight. Finally, the samples were centrifugated for 30 min at 25000 x g and 4°C before washing the resulting pellet with 1 ml of 70 % (v/v) cold ethanol. After removing the supernatant, the pellet was air dried on ice. The resulting RNA was then diluted in the desired amount of RNase-free water for further use
|
| Label |
Cy3
|
| Label protocol |
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.
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| |
|
| Hybridization protocol |
The labelled RNA was fragmented and hybridized as described by the manufacturer's instructions for Agilent one color microarrays with 0.6 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 intensities were normexp background substracted (offset=50) and quantile normalized.
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| |
|
| Submission date |
Jun 14, 2016 |
| Last update date |
Jun 15, 2016 |
| 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 |
GPL21131 |
| Series (1) |
| GSE83363 |
Awaking from dormancy: Resuscitation of chlorotic Synechocystis sp. PCC 6803 cells reveals a highly orchestrated process |
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