|
| Status |
Public on May 06, 2021 |
| Title |
Wild_type_1737-no_Mn-rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Wild type 1737-no Mn
|
| Organism |
Corynebacterium diphtheriae |
| Characteristics |
strain: 1737
genotype: wild type
|
| Growth protocol |
Wild-type C. diphtheriae or the mntR mutant were grown overnight in the semi-defined medium mPGT supplemented with 1 µM FeCl3. Overnight cultures were diluted 1:1 with fresh media and allowed to grow for 2-3 h. The optical density of cultures was measured (A600) and the cells diluted to an optical density of 0.1 into mPGT supplemented with 0.5 µM FeCl3 (required for growth) and either 0 or 5 µM MnCl2. Cells were harvested by centrifugation at mid-logarithmic growth.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lysed in phosphate buffered saline supplemented with 10% ethanol, 0.2% phenol, and 14.3 mM beta-mercaptoethanol in Lysing Matrix B (MP Biomedicals). Appropriate volumes of TriZOL LS reagent (Thermo Fisher Scientific) were added to lysates and samples were briefly vortexed. RNA was purified following the Zymo Direct-zol RNA MiniPrep Plus protocol (Zymo Research Corporation). RNA was treated with Ambion Turbo DNAse I (Invitrogen). The RNA concentration and integrity were assessed by the Agilent Bioanalyzer 2100 following the RNA 6000 Nano protocol.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 labeled cRNA was prepared from 0.1 µg RNA using the Low Input Quick Amp WT Labeling Kit (Agilent) following the manufacturer’s instructions. cRNA was purified using the RNAeasy kit (QIAGEN). Dye incorporation and cRNA yield were assessed with the GE NanoVue Plus.
|
| |
|
| Hybridization protocol |
0.6 µg of Cy3-labelled cRNA (specific activity > 30 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 m in a reaction volume of 25 µl. An equal volume (25 µl) of 2x Hi-RPM Hybridization Buffer was added to stop the fragmentation reaction. 40 µl of each sample was hybridized to Agilent Custom Microarrays designed using the Corynebacterium diphtheriae strain NCTC 13129 for 17 h at 65°C in a rotating hybridization oven. After hybridization, microarrays were washed 1 m at room temperature with Gene Expression Wash Buffer 1 and 1 m at 37°C using Gene Expression Wash Buffer 2. Slides were scanned immediately.
|
| Scan protocol |
Slides were scanned on the Agilent G2600D SureScan Microarray Scanner using settings recommended by the manufacturer (Dye channel: G (green), Scan region: Scan Area 61 x 21.6 mm, Scan Resolution: 3 µm, and 100% Green PMT gain).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software version 12.0.3.1 (Agilent) using default parameters to obtain gProcessedSignal values. Median gProcessedSignal values were divided by the entire sample set mean and log2 transformed.
|
| |
|
| Submission date |
May 05, 2021 |
| Last update date |
May 06, 2021 |
| Contact name |
Michael Schmitt |
| Organization name |
US Food and Drug Administration
|
| Street address |
10903 New Hampshire Ave
|
| City |
Silver Spring |
| State/province |
MD |
| ZIP/Postal code |
20993 |
| Country |
USA |
| |
|
| Platform ID |
GPL30087 |
| Series (1) |
| GSE173960 |
Identification of Corynebacterium diphtheriae manganese and MntR regulated genes |
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