|
| Status |
Public on May 17, 2021 |
| Title |
Array regA_aerobic_2 |
| Sample type |
RNA |
| |
|
| Source name |
Burkholderia pseudomallei DregA mutant
|
| Organism |
Burkholderia pseudomallei |
| Characteristics |
strain: E8
phenotype: avirulent
|
| Treatment protocol |
100 µg total RNA per sample were treated with DNA-freeTM Kit from Ambion (USA) according to the manufacturer's instructions to obtain DNA-free RNA. After precipitation of the RNA with ethanol and resuspension in nuclease free water, 10 µg per sample were subjected to ribosomal RNA depletion using the MICROBExpressTM kit from Ambion (USA) according to the manufacturer's instructions. Afterwards, 2 µg of purified mRNA were used for cDNA synthesis utilizing Superscript II Reverse Transcriptase (Life Technologies).
|
| Growth protocol |
B. pseudomallei was cultivated in 60 ml Lysogeny broth at 37 °C and 140 rpm OD650nm 0.5 under aerobic conditions. Aerobic samples (1-6) were taken at this time point. Samples 7-42 were also grown aerobically to OD650nm 0.5 but then either incubated anaerobically (tightly closed Falcon tubes) without nitrate (samples 7-12, 19-24, 31-36) or with 50 mM nitrate (samples 13-18, 24-30, 37-42) for 30 min under various agitation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
50 ml of cell suspension were harvested and centrifuged at 2°C at 10,000 g for 5 min. The cell pellets were quickly resuspended in 1 ml of Trizol Reagent (Invitrogen). After the RNA extraction procedure according to manufacturer's instructions, the integrity of the RNA was assessed by agarose gel electrophoresis and tested for the absence of DNA contamination by PCR.
|
| Label |
Cy5
|
| Label protocol |
1.5 µg of obtained cDNA were used for the labeling procedure using the ULSTM arrayCGH Labeling Kit (Leica Microsystem) according to the manufacturer´s instructions.
|
| |
|
| Hybridization protocol |
Microarray hybridizations were performed at 65°C in a MAUI hybridization station (BioMicro Systems, USA) for 17hrs.
|
| Scan protocol |
The slides were washed, dried in a centrifuge at 500rpm for 2mins, and scanned on an Axon Genepix 4000B scanner (Molecular Devices, USA) at a resolution of 5 microns
|
| Description |
This sample is of DregA mutant of Burkholderia pseudomallei E8 and the one of three DregA mutant biological replicates used in this experiment, each from separate cultures. Aerobic growth condition.
|
| Data processing |
The raw data (.pair file) was subjected to Robust Multi-array Average (RMA) procedure as implemented in Roche NimbleGen (USA) and normalized using the quantile normalization method developed from Bolstad et al. (2005) (Bolstad BM, Irizarry RA, Astrand M, Speed TP (2003). "A comparison of normalization methods for high density oligonucleotide array data based on variance and bias". Bioinformatics. 19 (2): 185–93.
processed data: RMA-normalized, averaged gene-level signal intensity
|
| |
|
| Submission date |
Oct 19, 2020 |
| Last update date |
May 17, 2021 |
| Contact name |
Christian Kohler |
| E-mail(s) |
christian.kohler@med.uni-greifswald.de
|
| Organization name |
University Medicine Greifswald
|
| Department |
Friedrich Loeffler Institue of Medical Microbiology
|
| Street address |
Ferdinand Sauerbruch Strasse 1
|
| City |
Greifswald |
| ZIP/Postal code |
17489 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16432 |
| Series (1) |
| GSE159521 |
RegAB Two-Component System Homolog of Burkholderia pseudomallei is the Master Regulator of Redox Control and involved in Virulence. |
|
