|
| Status |
Public on Jun 03, 2011 |
| Title |
∆idr1∆idr2 double mutant 20 minutes after addition of iron, replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Halobacterium salinarum, iron
|
| Organism |
Halobacterium salinarum NRC-1 |
| Characteristics |
strain: ∆idr1∆idr2
iron concentration (um): 100
time (min): 20
|
| Treatment protocol |
Cultures were split in half and FeSO4 was added to one half, while the other was continued under iron limitation. 8-mL samples were collected from each culture every 20 minutes for 60 minutes (see also experimental design, Supplementary Figure 1). The zero time point was harvested immediately before the addition of iron. At each time point, samples were removed from the culture, pelleted for 30 seconds at 15 krpm in a microcentrifuge and snap frozen in liquid nitrogen. Pellets were stored at -80°C overnight
|
| Growth protocol |
The Δura3 parent, Δidr2 and Δidr1, and Δ idr1Δidr2 mutant strains were grown to mid-logarithmic phase (OD600 ~0.4 – 0.8) in CDM (Schmid et al., 2009, Mol Sys Biol, 5:282) with all trace metals except iron.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared the following day using the Absolutely RNA miniprep kit according to manufacturer’s instructions (Agilent Technologies, Santa Clara, CA). RNA quality was measured using an Agilent Bioanalyzer as described (Schmid et al., 2009). Freedom from DNA contamination was determined on 200 ng samples of RNA in 25 cycles of PCR.
|
| Label |
cy3, cy5
|
| Label protocol |
5 ug quality-checked total RNA was direct-labeled with Cy3 and Cy5 dyes according to manufacturer's instructions (Kreatech, Alameda, CA).
|
| |
|
| Channel 2 |
| Source name |
Halobacterium standard reference sample
|
| Organism |
Halobacterium salinarum NRC-1 |
| Characteristics |
strain: NRC-1
iron concentration (um): 0
time (min): NA
|
| Treatment protocol |
Standard mid-log phase (OD600 - 0.5-0.6, 37C, 225 rpm) Halobacterium salinarum reference sample.
|
| Growth protocol |
Standard mid-log phase (OD600 - 0.5-0.6, 37C, 225 rpm) Halobacterium salinarum reference sample.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared the following day using the Absolutely RNA miniprep kit according to manufacturer’s instructions (Agilent Technologies, Santa Clara, CA). RNA quality was measured using an Agilent Bioanalyzer as described (Schmid et al., 2009). Freedom from DNA contamination was determined on 200 ng samples of RNA in 25 cycles of PCR.
|
| Label |
cy5, cy3
|
| Label protocol |
5 ug quality-checked total RNA was direct-labeled with Cy3 and Cy5 dyes according to manufacturer's instructions (Kreatech, Alameda, CA).
|
| |
|
| |
| Hybridization protocol |
see Baliga NS, Bjork SJ, Bonneau R, Pan M, Iloanusi C, Kottemann MC, Hood L, DiRuggiero J. Systems level insights into the stress response to UV radiation in the halophilic archaeon Halobacterium NRC-1.Genome Res. 2004 Jun;14(6):1025-35
|
| Scan protocol |
ScanArray 5000, using Packard BioChip ScanArray software. See http://www.systemsbiology.org/Scientists_and_Research/Technology/ISB_Facilities/Microarray for microarray scanning details
|
| Description |
raw data file 13112.csv: Halobacterium salinarum, iron (ch1) Sample is labeled with Cy3
raw data file 13113.csv: Halobacterium standard reference sample (ch1) Sample is labeled with Cy3
|
| Data processing |
AnalyzerDG software for spotfinding. Intensities were normalized between the Alexa 546 and Alexa 647 channels by scaling all intensities in one channel such that the 75th percentile in each channel was made equal. See http://www.systemsbiology.org/Scientists_and_Research/Technology/Data_Management/Microarray_Pipeline for more details.
VERA/SAM normalized log10 ratio (test/reference); see also: Marzolf B, Deutsch EW, Moss P, Campbell D, Johnson MH, Galitski T. 2006. SBEAMS-Microarray: database software supporting genomic expression analyses for systems biology. BMC Bioinformatics. 7:286.
Each Sample is based on two arrrays (one with dye-swap).
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| |
|
| Submission date |
Jun 02, 2011 |
| Last update date |
Jun 03, 2011 |
| Contact name |
Amy K Schmid |
| E-mail(s) |
amy.schmid@duke.edu
|
| Organization name |
Duke University
|
| Department |
Biology
|
| Lab |
Schmid
|
| Street address |
125 Science Dr.
|
| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27707 |
| Country |
USA |
| |
|
| Platform ID |
GPL13682 |
| Series (2) |
| GSE29704 |
Two transcription factors are necessary for iron homeostasis in a salt-dwelling archaeon [gene expression data] |
| GSE29706 |
Two transcription factors are necessary for iron homeostasis in a salt-dwelling archaeon |
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