|
| Status |
Public on Jul 31, 2018 |
| Title |
sigD_overexpress_4th |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
WT carrying the sigD overexpressing plasmid before IPTG induction
|
| Organism |
Corynebacterium glutamicum R |
| Characteristics |
genotype/variation: carrying the sigD overexpressing plasmid in WT background
plasmid: pCRC646
time point: before IPTG induction
|
| Treatment protocol |
To induce sigD overexpression, isopropyl-β-d-thiogalactopyranoside (IPTG) was added at the final concentration of 0.5 mM to exponentially-growing culture. After 15 min, 1 ml of culture was mixed with 1 ml bacterial RNA protect reagent (Qiagen) for RNA stabilization.
|
| Growth protocol |
C. glutamicum strains are grown in 100 ml of nutrient rich A medium containing 1% glucose in 500 ml flask at 33˚C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from C. glutamicum cells using NucleoSpin RNA II kit (Macherey-Nagel, Germany) according to the manufacture's manual.
|
| Label |
Cy5
|
| Label protocol |
The cDNAs were synthesized from RNA by using reverse transcriptase from 10 μg of total RNAs and labeled with cyanine 3 (Cy3) or cyanine 5 (Cy5) using the SuperScript Indirect cDNA Labeling system (Life Technologies).
|
| |
|
| Channel 2 |
| Source name |
WT carrying the sigD overexpressing plasmid after 15 min of IPTG induction
|
| Organism |
Corynebacterium glutamicum R |
| Characteristics |
genotype/variation: carrying the sigD overexpressing plasmid in WT background
plasmid: pCRC646
time point: after 15 min of IPTG induction
|
| Treatment protocol |
To induce sigD overexpression, isopropyl-β-d-thiogalactopyranoside (IPTG) was added at the final concentration of 0.5 mM to exponentially-growing culture. After 15 min, 1 ml of culture was mixed with 1 ml bacterial RNA protect reagent (Qiagen) for RNA stabilization.
|
| Growth protocol |
C. glutamicum strains are grown in 100 ml of nutrient rich A medium containing 1% glucose in 500 ml flask at 33˚C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from C. glutamicum cells using NucleoSpin RNA II kit (Macherey-Nagel, Germany) according to the manufacture's manual.
|
| Label |
Cy3
|
| Label protocol |
The cDNAs were synthesized from RNA by using reverse transcriptase from 10 μg of total RNAs and labeled with cyanine 3 (Cy3) or cyanine 5 (Cy5) using the SuperScript Indirect cDNA Labeling system (Life Technologies).
|
| |
|
| |
| Hybridization protocol |
Labeled cDNA (4.5 μg each) are mixed with 1 × hybridization buffer (6 × SSC, 0.2% SDS, 5 × Denhardt’s solution, 0.1 mg/ml denatured salmon sperm DNA) in a total volume of 340 μl, and denatured at 95˚C for 2 min. The hybridization mixture was hybridized to a microarray in an Agilent Technologies Microarray chamber at 60˚C for 17 h in a rotating Agilent hybridization oven. After hybridization, microarrays were washed for 5 minutes with wash buffer 1 (2x SSC, 0.2% SDS) at room temperature , 5 minutes with wash buffer 2 (0.2x SSC, 0.2% SDS) at 60˚C, and 5 minutes with wash buffer 3 (0.2x SSC) at room temperature.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) at a resolution of 5 µm. PMT is set to 100% for Cy3 and Cy5 channels.
|
| Description |
sigD_ox_4th
IPTG was added at the final concentration of 0.5 mM during the exponential phase of growth
|
| Data processing |
The scanned images were analyzed quantified with Feature Extraction Software 10.5.5.1 (Agilent) using default parameters (protocol GE2-NonAT_105_Dec08). Feature extracted data were analyzed using GeneSpring GX v 12.5 software from Agilent. Normalization of the data was done in GeneSpring GX using the Lowess normalization algorithm without baseline transformation.
|
| |
|
| Submission date |
Aug 07, 2017 |
| Last update date |
Jul 31, 2018 |
| Contact name |
Masayuki Inui |
| E-mail(s) |
mmg-lab@rite.or.jp
|
| Organization name |
Research institute of Innovative Technology for the Earth (RITE)
|
| Street address |
9-2 Kizugawadai, Kizugawa
|
| City |
Kyoto |
| ZIP/Postal code |
619-0292 |
| Country |
Japan |
| |
|
| Platform ID |
GPL20864 |
| Series (2) |
| GSE102324 |
Overexpression of sigD in the wild type |
| GSE102329 |
Genome-wide analyses of C. glutamicum SigD |
|
