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Sample GSM2734735 Query DataSets for GSM2734735
Status Public on Jul 31, 2018
Title sigD_overexpress_3rd
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_3rd
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) [email protected]
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

Data table header descriptions
ID_REF
VALUE Normalized log2 ratio (Cy5/Cy3)

Data table
ID_REF VALUE
ID_71 0.4846182
ID_451 -1.9487076
ID_831 -0.49898052
ID_1211 -0.76171684
ID_1591 -0.14800358
ID_1971 -0.37996387
ID_2351 0.1579814
ID_2731 0.28534126
ID_3111 0.38144207
ID_1229 -0.6843395
ID_2850 0.53527546
ID_65 -0.13330507
ID_445 0.12007761
ID_825 0.6333666
ID_1205 0.04788971
ID_1585 0.017269135
ID_1965 0.26958466
ID_2345 -0.2755332
ID_2725 0.2673397
ID_3105 0.26473808

Total number of rows: 3282

Table truncated, full table size 60 Kbytes.




Supplementary file Size Download File type/resource
GSM2734735_sigD_ox_3rd.txt.gz 639.4 Kb (ftp)(http) TXT
Processed data included within Sample table

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