|
| Status |
Public on Aug 13, 2012 |
| Title |
∆VNG0258 mutant, immediately before the addition of H2O2, replicate B |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
∆VNG0258 mutant immediately before the addition of H2O2
|
| Organism |
Halobacterium salinarum NRC-1 |
| Characteristics |
strain: ∆VNG0258 mutant
[h2o2]: 0 mM
[paraquat]: 0 mM
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For each biological duplicate time course, all samples were removed from the same culture to ensure coherence of gene expression between unstressed and stressed cultures. From each sample, cells were immediately pelleted by centrifugation (12,000g, 30 sec, 25°C) and snap-frozen in liquid nitrogen. Sample pellets were stored overnight at -80C, followed by RNA preparation using the Absolutely-RNA kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. RNA quality was assessed using the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Freedom from DNA contamination was ensured by PCR amplification of 200 ng of each RNA sample.
|
| Label |
cy5
|
| Label protocol |
600 ng of each quality-checked RNA sample directly labeled with Cy3 and Cy5 dyes (Kreatech) as described previously (Schmid et. al, 2007) and combined in equimolar amounts with oppositely labeled H. salinarum NRC-1 reference RNA (from batch cultures grown in CM at 37C to mid-logarithmic phase). This common reference RNA was used across all ~950 microarray experiments listed in the H. salinarum NRC-1 microarray data repository.
|
| |
|
| Channel 2 |
| Source name |
Halobacterium standard reference sample
|
| Organism |
Halobacterium salinarum NRC-1 |
| Characteristics |
strain: NRC-1 wild type
[h2o2]: 0 mM
[paraquat]: 0 mM
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For each biological duplicate time course, all samples were removed from the same culture to ensure coherence of gene expression between unstressed and stressed cultures. From each sample, cells were immediately pelleted by centrifugation (12,000g, 30 sec, 25°C) and snap-frozen in liquid nitrogen. Sample pellets were stored overnight at -80C, followed by RNA preparation using the Absolutely-RNA kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. RNA quality was assessed using the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Freedom from DNA contamination was ensured by PCR amplification of 200 ng of each RNA sample.
|
| Label |
cy3
|
| Label protocol |
600 ng of each quality-checked RNA sample directly labeled with Cy3 and Cy5 dyes (Kreatech) as described previously (Schmid et. al, 2007) and combined in equimolar amounts with oppositely labeled H. salinarum NRC-1 reference RNA (from batch cultures grown in CM at 37C to mid-logarithmic phase). This common reference RNA was used across all ~950 microarray experiments listed in the H. salinarum NRC-1 microarray data repository.
|
| |
|
| |
| Hybridization protocol |
Samples were hybridized to a custom 60-mer oligonucleotide microarray (Agilent technologies, Santa Clara, CA, 8 x 15,000 feature array, AMADID ID #30108). Probes for each ORF were spotted on each array six-fold and dye-swapping was conducted (to rule out bias in dye incorporation) for all samples, yielding 12 technical replicates per gene per time point. Slide hybridization and washing protocols were performed according to the manufacturer’s instructions except that hybridization was conducted in the presence of 37.5% formamide at 68˚C to ensure proper stringency due to the high G+C content of the H. salinarum genome.
|
| Scan protocol |
Slide scanning and spotfinding were conducted using Feature Extraction software (Agilent).
|
| Description |
H2O2_d258_0000min_vs_NRC1_B
|
| Data processing |
Using the R/Bioconductor m-array and limma packages, resultant raw data were background-subtracted using normexp (Silver JD, Ritchie ME, and Smyth GK, 2007) Loess normalized within each array, and quantile normalized between all arrays. Any of the 12 gene-specific probes for each gene lying outside the 99th% confidence interval were removed using Dixon’s test (Dixon, 1951). Finally, remaining probe intensities for each gene were averaged and log2 ratios were calculated, yielding one expression ratio per gene. Note that the values here are the averages for the forward array and dye flip array individually after the outliers have been removed. As such, their average needs to be weighted by the number of probes from which the average was calculated (either 5 or 6 dependent on whether an outlier was removed).
|
| |
|
| Submission date |
Nov 28, 2011 |
| Last update date |
Aug 13, 2012 |
| Contact name |
Nicholas A Gillum |
| E-mail(s) |
nicholas.gillum@duke.edu
|
| Phone |
508-769-1267
|
| Organization name |
Duke University
|
| Street address |
Box 97925
|
| City |
Durham |
| State/province |
North Carolina |
| ZIP/Postal code |
27708 |
| Country |
USA |
| |
|
| Platform ID |
GPL14876 |
| Series (1) |
| GSE33980 |
RosR is a haloarchaeal-specific transcription factor required for the response to extreme oxidative stress in Halobacterium salinarum NRC-1. |
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