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Sample GSM839967 Query DataSets for GSM839967
Status Public on Aug 13, 2012
Title ∆ura3 strain, 80 minutes after the addition of paraquat, replicate A, dye flip
Sample type RNA
 
Channel 1
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 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 ∆ura3 strain_80 min after the addition of paraquat
Organism Halobacterium salinarum NRC-1
Characteristics strain: ∆ura3 isogenic parent control
[h2o2]: 0 mM
[paraquat]: 0.333 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 PQ_NRC1_vs_ura3_0080min_A
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.

Data table header descriptions
ID_REF
VALUE Loess normalized log2 expression ratio (test/reference)

Data table
ID_REF VALUE
VNG0001H 0.307002925
VNG0002G 0.236853523
VNG0003C -0.122810635
VNG0005H -0.254957963
VNG0006G -0.366452665
VNG0008G 0.2793847
VNG0009G 0.256939101
VNG0011C -0.172534869
VNG0013C 0.40513469
VNG0014C 0.207300505
VNG0015H 0.381772893
VNG0016H -0.173355652
VNG0017H 0.165574865
VNG0018H 0.247969244
VNG0019H 0.080797525
VNG0020H -0.217416314
VNG0021H -0.05584981
VNG0022H -0.445007087
VNG0023H -0.630535236
VNG0024H -0.422327661

Total number of rows: 2410

Table truncated, full table size 50 Kbytes.




Supplementary file Size Download File type/resource
GSM839967_253010810020_S01_GE2-v5_95_Feb07_2_1.txt.gz 4.3 Mb (ftp)(http) TXT
Processed data included within Sample table
Processed data are available on Series record

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