|
| Status |
Public on Apr 02, 2013 |
| Title |
PCC6803_WT_CT23.5_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
30 minutes before light onset
|
| Organism |
Synechocystis sp. PCC 6803 |
| Characteristics |
genotype: wildtype
|
| Treatment protocol |
The optical density of the culture was monitored by measuring the absorbance at 750 nm. Cultures were synchronized with three cycles of light/dark 12h:12h prior sampling. Aliquots were taken at OD750 ≈ 0.5.
|
| Growth protocol |
Liquid cultures of the Synechocystis 6803 WT strain were grown in BG11 medium (Rippka et al., 1979) at 30°C under continuous illumination with white light at 120 µmol photons•m–2•s– 1 and a continuous stream of air.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Over a 24 h time course, 6 samples for RNA isolation were taken at the following time points: 30 minutes before and after light is switched off (sample 1 - CT 11.5 and sample 2 - CT 12.5), 30 minutes before midnight (sample 3 - CT 17.5), 348 30 minutes before and after light onset (sample 4 - CT 23.5 and sample 5 - CT 0.5) and 30 minutes before noon (sample 6 - CT 5.5). Cells were filtered rapidly through Supor 0.45µm membrane filters (PALL), immediately stowed with TRIzol reagent (Invitrogen) and frozen in liquid nitrogen. Total RNA samples stored at −20°C were transferred directly to a 65°C waterbath for 5 minutes, mixed with 0.2 ml chloroform per ml of TRIzol and incubated for 15 minutes. The dissolving of the membrane and lyses of the cells were supported by vortexing. Centrifugation at maximum speed for 10 min at 4°C separated the phases. The RNA in the supernatant was precipitated by adding 0.5 ml of isopropanol per ml TRIzol used in the initial homogenisation.
|
| Label |
Cy3
|
| Label protocol |
The 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 1.5 µg of labeled RNA
|
| Scan protocol |
Arrays were scanned on the Agilent Technologies Scanner G2505B, 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 using R language. Signal intensity was probe-wise LOS normalized, using a oscillation threshold of 0.7.
|
| |
|
| Submission date |
Apr 01, 2013 |
| Last update date |
Apr 02, 2013 |
| Contact name |
Robert Lehmann |
| E-mail(s) |
[email protected]
|
| Organization name |
Humboldt University Berlin
|
| Department |
Institute for Theoretical Biology
|
| Street address |
Invalidenstrasse 43
|
| City |
Berlin |
| ZIP/Postal code |
10115 |
| Country |
Germany |
| |
|
| Platform ID |
GPL15867 |
| Series (1) |
| GSE45667 |
How cyanobacteria pose new problems to old methods: Challenges in microarray time series analysis |
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