|
| Status |
Public on May 30, 2013 |
| Title |
LDDDD_CT0.5_day3 |
| Sample type |
RNA |
| |
|
| Source name |
whole cell
|
| Organism |
Synechocystis sp. PCC 6803 |
| Characteristics |
light regime: LDDDD
sample time point: CT0.5
|
| Treatment protocol |
Cell concentrations were measured by determining the optical densities of the culture at 750 nm (OD750). Cultures were synchronized with three cycles of light/dark 12h:12h prior sampling.
|
| Growth protocol |
The glucose-tolerant and motile wild-type strain of Synechocystis sp. PCC 6803 originally obtained from S. Shestakov (Moscow State University, Russia) was routinely grown photoautotrophically in BG11-medium at 30°C under continuous illumination with white light of 80 µmol of photons/m^2s and a continuous stream of air.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
15 ml of cyanobacterial culture were filtered rapidly through Supor 0.45 µm 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 water bath for 5 min, mixed with 0.2 ml chloroform per ml of TRIzol and incubated for further 15 min. 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 1 volume of isopropanol. Finally isolated total RNA was treated with RNase-free Turbo™DNase (Ambion) by following the manufacturer's instructions, resulting in 40 µl purified RNA solution.
|
| 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.
|
| Description |
subjective day
|
| Data processing |
Raw data were processed using GNU R and MATLAB. Signal intensity was probe-wise LOS normalized, using a oscillation threshold of 0.7.
|
| |
|
| Submission date |
May 29, 2013 |
| Last update date |
May 30, 2013 |
| Contact name |
Christian Johannes Beck |
| Organization name |
Humboldt-University Berlin
|
| Department |
Institute for Theoretical Biology
|
| Lab |
AG Axmann
|
| Street address |
Invalidenstrasse 43
|
| City |
Berlin |
| State/province |
Berlin |
| ZIP/Postal code |
10115 |
| Country |
Germany |
| |
|
| Platform ID |
GPL15867 |
| Series (1) |
| GSE47482 |
A daily temporal program for rhythmic expression of protein-coding and non-coding genes in response to light and dark in Synechocystis sp. PCC 6803 |
|
