|
| Status |
Public on May 13, 2019 |
| Title |
pdxJ_11plus_vs_pdxJ_11minus_I |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
pdxJ(11+)
|
| Organism |
Escherichia coli BW25113 |
| Characteristics |
genotype/variation: pdxJ(11+)
sample type: sample
|
| Growth protocol |
Strains were grown in M9-glucose minimal medium (1x M9-salts, 2 mM MgSO4, 0.1 mM CaCl2, 0.4 % Glucose). When the culture reached an optical density of 0.5 cells were harvested by centrifugation(3 min, 5000 x g) mixed with crushed ice to ensure rapid cooling. The pelleted cells were shock-frozen in liquid nitrogen and stored at -80° C until RNA preparation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The preparation of total RNA was performed as described previously with the RNeasy Kit from Qiagen (Kranz et al., 2018, BMC Genomics 19(1):753; doi: 10.1186/s12864-018-5111-1).
|
| Label |
Cy5
|
| Label protocol |
Synthesis of fluorescently labelled cDNA were carried out as described (Kranz et al., 2018, BMC Genomics 19(1):753; doi: 10.1186/s12864-018-5111-1).
|
| |
|
| Channel 2 |
| Source name |
pdxJ(11-)
|
| Organism |
Escherichia coli BW25113 |
| Characteristics |
genotype/variation: pdxJ(11-)
sample type: control
|
| Growth protocol |
Strains were grown in M9-glucose minimal medium (1x M9-salts, 2 mM MgSO4, 0.1 mM CaCl2, 0.4 % Glucose). When the culture reached an optical density of 0.5 cells were harvested by centrifugation(3 min, 5000 x g) mixed with crushed ice to ensure rapid cooling. The pelleted cells were shock-frozen in liquid nitrogen and stored at -80° C until RNA preparation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The preparation of total RNA was performed as described previously with the RNeasy Kit from Qiagen (Kranz et al., 2018, BMC Genomics 19(1):753; doi: 10.1186/s12864-018-5111-1).
|
| Label |
Cy3
|
| Label protocol |
Synthesis of fluorescently labelled cDNA were carried out as described (Kranz et al., 2018, BMC Genomics 19(1):753; doi: 10.1186/s12864-018-5111-1).
|
| |
|
| |
| Hybridization protocol |
Purified cDNA samples to be compared were pooled and prepared using Agilent’s Gene Expression Hybridization Kit. The two-color samples were hybridized at 65°C while rotating for 17 hours using Agilent's hybridization chamber and hybridization oven. After hybridization the arrays were washed using Agilent’s Wash Buffer Kit according to the manufacturer’s instructions.
|
| Scan protocol |
Fluorescence of hybridized DNA microarrays was determined at 532 nm (Cy3) and 635 nm (Cy5) at 5 μm resolution with a GenePix 4000B laser scanner and GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, CA, USA). Fluorescence images were saved to raw data files in TIFF format (GenePix Pro 6.0). Quantitative TIFF image analysis was carried out using GenePix image analysis software and results were saved as GPR-file (GenePix Pro 6.0).
|
| Description |
Ratio (pdxJ(11+) / pdxJ(11-))
|
| Data processing |
For ratio calculation and ratio normalization, GPR-files were processed using the BioConductor R-packages limma and marray (http://www.bioconductor.org).
|
| |
|
| Submission date |
Feb 07, 2019 |
| Last update date |
May 13, 2019 |
| Contact name |
Tino Polen |
| E-mail(s) |
t.polen@fz-juelich.de
|
| Organization name |
Forschungszentrum Jülich GmbH
|
| Department |
IBG-1: Biotechnology
|
| Street address |
Leo Brandt Str.
|
| City |
Juelich |
| State/province |
NRW |
| ZIP/Postal code |
52425 |
| Country |
Germany |
| |
|
| Platform ID |
GPL23003 |
| Series (1) |
| GSE126228 |
Comparison of expressed pdxJ variants on global gene expression in E. coli |
|
