|
| Status |
Public on Apr 01, 2012 |
| Title |
Control - Glucose vs. Glucose Replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Mid-exponential phase cultures
|
| Organism |
Rhodopirellula baltica |
| Characteristics |
strain: SH1T
growth condition: glucose
|
| Growth protocol |
R. baltica SH1T was grown on marine mineral medium, supplemented with glucose as reference substrate or substrates of interest. Cultures have been grown to mid-exponential before being harvested. Collected cell material was shock-frozen and kept at -80 °C until being further processed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Ambion's TRI reagent and mechanical lysis following standard procedures.
|
| Label |
Alexa546
|
| Label protocol |
5-10 µg of total RNA have been reverse transcribed using the SuperScript Direct cDNA labelling core kit (Applied Biosystems) following the manufacturers instructions. cDNA was purified using the QUIAEX II Gel extraction kit (Quiagen) according to the instructions described in the manual. Samples have been labelled applying the Platinum BrightTm Alexa 546 and 647 (Kreatech) nucleic acid labeling kits according to the manufacturers protocol.
|
| |
|
| Channel 2 |
| Source name |
Mid-exponential phase cultures
|
| Organism |
Rhodopirellula baltica |
| Characteristics |
strain: SH1T
growth condition: glucose
|
| Growth protocol |
R. baltica SH1T was grown on marine mineral medium, supplemented with glucose as reference substrate or substrates of interest. Cultures have been grown to mid-exponential before being harvested. Collected cell material was shock-frozen and kept at -80 °C until being further processed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Ambion's TRI reagent and mechanical lysis following standard procedures.
|
| Label |
Alexa647
|
| Label protocol |
5-10 µg of total RNA have been reverse transcribed using the SuperScript Direct cDNA labelling core kit (Applied Biosystems) following the manufacturers instructions. cDNA was purified using the QUIAEX II Gel extraction kit (Quiagen) according to the instructions described in the manual. Samples have been labelled applying the Platinum BrightTm Alexa 546 and 647 (Kreatech) nucleic acid labeling kits according to the manufacturers protocol.
|
| |
|
| |
| Hybridization protocol |
The hybridisation reaction inlcuding denaturing, hybridisation, washing and drying was done in a HS400 Pro Hybridisation station controlled by respective software (Tecan). Arrays have been blocked by pre-hybridisation buffer and blocking reagent. Differently labelled cDNA was pooled in DIG Easy Hyb hybridisation solution (Roche). After blocking, sample solutions were applied to the arrays followed by denaturation and hybridisation at stringent conditions for more than 12 hrs. ULTRArray low stringency wash buffer (Applied Biosystems) was used for final washing followed by drying.
|
| Scan protocol |
Scanner: ScanArray Express MicroArray scanner (Perkin Elmer)
Images were quantified using ScanArray Express Software (version 4.0)
|
| Description |
Control - Biological Replicate 2
|
| Data processing |
LOWESS normalisation, MADA software was used (http://www.mpi-bremen.de/en/MADA), for downstream data processing MayDay was applied (http://www-ps.informatik.uni-tuebingen.de/mayday/wp/)
|
| |
|
| Submission date |
Feb 15, 2012 |
| Last update date |
Apr 01, 2012 |
| Contact name |
Carl-Eric Wegner |
| E-mail(s) |
carl-eric.wegner@mpi-marburg.mpg.de
|
| Organization name |
Max Planck Institute for Terrestrial Microbiology
|
| Street address |
Karl von Frisch Straße 10
|
| City |
Marburg (Lahn) |
| ZIP/Postal code |
35043 |
| Country |
Germany |
| |
|
| Platform ID |
GPL7654 |
| Series (1) |
| GSE35832 |
Combining transcriptomics with functional phylogenomics elucidates the ecophysiology of sulfatases in marine bacteria |
|
