|
| Status |
Public on Feb 10, 2017 |
| Title |
B. subtilis PG479 Day 5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
B. subtilis PG479 Day 5
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: PG479
time (day): 5
|
| Treatment protocol |
To construct B. subtilis strain PG479 (kre::erm), kre was replaced with an erythromycin resistance cassette amplified from pMutin4. Competent B. subtilis cells were transformed directly with the ligation products and mutants were verified with PCR.
|
| Growth protocol |
B. subtilis strains were grown in LB at 37°C in the presence of 1.5% xylose and 1 mM IPTG (inducers were added in all cultures because some of the strains tested in the same experiment required induction). Cells were harvested at O.D.600 0.4-0.6.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
To isolate RNA, cell pellets were flash frozen in liquid nitrogen immediately after harvesting and stored at -80°C. Frozen pellets were grounded and subjected to RNA extraction
|
| Label |
Cy3
|
| Label protocol |
Labeling was performed by reverse transcription using random octamers, incorporating Cy3 for the test samples and Cy5 for the common reference
|
| |
|
| Channel 2 |
| Source name |
Pool of different samples of different B. subtilis strains
|
| Organism |
Bacillus subtilis |
| Characteristics |
sample type: Pool of different samples of different B. subtilis strains
|
| Treatment protocol |
No specific treatment was applied.
|
| Growth protocol |
B. subtilis strains were grown in LB at 37°C in the presence of 1.5% xylose and 1 mM IPTG (inducers were added in all cultures because some of the strains tested in the same experiment required induction). Cells were harvested at O.D.600 0.4-0.6.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
To isolate RNA, cell pellets were flash frozen in liquid nitrogen immediately after harvesting and stored at -80°C. Frozen pellets were grounded and subjected to RNA extraction
|
| Label |
Cy5
|
| Label protocol |
Labeling was performed by reverse transcription using random octamers, incorporating Cy3 for the test samples and Cy5 for the common reference
|
| |
|
| |
| Hybridization protocol |
Hybridization, washing, and scanning was performed as described in the Two-Color Microarray-Based Gene Expression Analysis manual (Version 6.6, Agilent Technologies). Briefly, hybridization mixtures were made by combining 300 ng test (Cy3) and 300 ng common reference (Cy5) material and were subsequently hybridized to the Agilent SurePrint Custom 8x15k microarrays G2509F (Agilent Technologies).
|
| Scan protocol |
The slides were scanned with the Agilent G2505C scanner as described in the Agilent Two-Color Microarray-Based Gene Expression Analysis guide (G4140-90050, Agilent technologies).
|
| Data processing |
Data were extracted with Feature Extraction software (Agilent Technologies). The data were analyzed in R-2.14.1 (http://cran.r-project.org/). All arrays were subjected to a set of quality control checks, such as visual inspection of the scans, checking for spatial effects through pseudo-color plots, and inspection of pre- and post-normalized data with box plots, density plots, ratio-intensity plots and principal component analysis. The data from B. subtilis strains PG479 and 168 were normalized, together with data from other strains, using the robust multi-array average (RMA) algorithm (Irizarry et al.).
|
| |
|
| Submission date |
Sep 25, 2014 |
| Last update date |
Feb 10, 2017 |
| Contact name |
Martijs Jonker |
| E-mail(s) |
m.j.jonker@uva.nl
|
| Organization name |
University of Amsterdam
|
| Department |
Microarray Department
|
| Street address |
Science Park 904
|
| City |
Amsterdam |
| ZIP/Postal code |
1090 GE |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL19229 |
| Series (1) |
| GSE61757 |
A novel feedback loop that controls bimodal expression of genetic competence |
|
