|
| Status |
Public on Sep 14, 2016 |
| Title |
3_cgpS_N_100_IPTG |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
C. glutamicum pAN6 cgpS-N
|
| Organism |
Corynebacterium glutamicum |
| Characteristics |
growth phase: exponential
|
| Treatment protocol |
CGXII medium + 2 % glucose, 100 µM IPTG, 30°C
|
| Growth protocol |
The transcriptome of C. glutamicum, in which the CgpS protein was downregulated by countersilencing, was compared with the untreated transcriptome of C. glutamicum ATCC 13032 carrying the empty plasmid. Both strains were grown in defined minimal medium CGXII + 2 % glucose as sole carbon source and cultivated at 30°C. Cells were harvested at an OD600 of 5 in pre-cooled ice-filled tubes via centrifugation (4,500 x g, 10 min, and 4 °C) and subsequently frozen in liquid nitrogen as well as stored at -70°C until preparation of total RNA.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The preparation of total RNA was performed as described elsewhere with the RNeasy Kit from Qiagen (Polen T, Schluesener D, Poetsch A, Bott M, Wendisch VF (2007) Characterization of citrate utilization in Corynebacterium glutamicum by transcriptome and proteome analysis. FEMS Microbiol Lett. 273(1):109-19).
|
| Label |
Cy5
|
| Label protocol |
Synthesis of fluorescently labelled cDNA were carried out as described elsewhere (Polen T, Schluesener D, Poetsch A, Bott M, Wendisch VF (2007) Characterization of citrate utilization in Corynebacterium glutamicum by transcriptome and proteome analysis. FEMS Microbiol Lett. 273(1):109-19).
|
| |
|
| Channel 2 |
| Source name |
C. glutamicum empty plasmid
|
| Organism |
Corynebacterium glutamicum |
| Characteristics |
growth phase: exponential
|
| Treatment protocol |
CGXII medium + 2 % glucose, 100 µM IPTG, 30°C
|
| Growth protocol |
The transcriptome of C. glutamicum, in which the CgpS protein was downregulated by countersilencing, was compared with the untreated transcriptome of C. glutamicum ATCC 13032 carrying the empty plasmid. Both strains were grown in defined minimal medium CGXII + 2 % glucose as sole carbon source and cultivated at 30°C. Cells were harvested at an OD600 of 5 in pre-cooled ice-filled tubes via centrifugation (4,500 x g, 10 min, and 4 °C) and subsequently frozen in liquid nitrogen as well as stored at -70°C until preparation of total RNA.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The preparation of total RNA was performed as described elsewhere with the RNeasy Kit from Qiagen (Polen T, Schluesener D, Poetsch A, Bott M, Wendisch VF (2007) Characterization of citrate utilization in Corynebacterium glutamicum by transcriptome and proteome analysis. FEMS Microbiol Lett. 273(1):109-19).
|
| Label |
Cy3
|
| Label protocol |
Synthesis of fluorescently labelled cDNA were carried out as described elsewhere (Polen T, Schluesener D, Poetsch A, Bott M, Wendisch VF (2007) Characterization of citrate utilization in Corynebacterium glutamicum by transcriptome and proteome analysis. FEMS Microbiol Lett. 273(1):109-19).
|
| |
|
| |
| Hybridization protocol |
Purified cDNA samples to be compared were pooled and the prepared two-color samples were hybridized at 65°C while rotating for 17 hours using Agilent’s Gene Expression Hybridization Kit, hybridization oven and hybridization chamber. After hybridization the arrays were washed using Agilent’s Wash Buffer Kit according to the manufacturer’s instructions.
|
| Scan protocol |
After washing the microarrays were dried by centrifugation (5 min; 1,600 x g) and fluorescence was determined at 532 nm (Cy3-dUTP) and 635 nm (Cy5-dUTP) with 5 µm resolution using an Axon GenePix 4000B laser scanner (Axon Instruments, Sunnyvale, U.S.A).
|
| Data processing |
Quantitative image analysis was carried out using GenePix image analysis software and results were saved as GPR-file (GenePix Pro 7.0, Axon Instruments). For data normalization, GPR-files were processed using the BioConductor/R-packages limma and marray (www.bioconductor.org). Processed and normalized data as well as experimental details (according to MIAME) were stored in the in-house microarray database for further analysis. For determination of differentially expressed genes data were quality filtered as decribed (Polen et al., 2007) using Signal/Noise >=5 for Cy5 or Cy3 channel.
|
| |
|
| Submission date |
Apr 26, 2016 |
| Last update date |
Apr 14, 2021 |
| 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 |
GPL20268 |
| Series (1) |
| GSE80674 |
Silencing of cryptic prophages in Corynebacterium glutamicum |
|
| Relations |
| Reanalyzed by |
GSM5197346 |
