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Sample GSM275403 Query DataSets for GSM275403
Status Public on May 13, 2008
Title evolved_temperature_Up_16min
Sample type mixed
 
Channel 1
Source name genomic DNA
Organism Escherichia coli K-12
Characteristics MG1655
Genomic DNA served as a universal reference
Treatment protocol Physiological perturbations were carried out under a controlled environment in the context of bioreactor (Bioflo 110, New Brunswick Scientific) growth. Thermoelectric sensors and heaters were used to shift temperature profiles between 250 C and 370 C, and polarographic dissolved oxygen sensors (Mettler Toledo) and nitrogen gas was used to rapidly change oxygen saturation between anaerobic (0% dissolved oxygen) and aerobic (16-21% dissolved oxygen) condition.
Growth protocol Bacteria were grown in batch or bioreactor vessels in M9 minimal media supplemented with 0.4% glucose.
Extracted molecule genomic DNA
Extraction protocol DNA extraction: Genomic DNA was isolated and purified according to standard procedures (Ausubel et al. 1994). Genomic DNA was fragmented by nebulization as described by Girgis et al. PLoS Genetics 3(9): e154 (2007) and purified by phenol/chloroform extraction and ethanol precipitated.
Label Cy5
Label protocol Fluorescent cDNA was synthesized from total RNA with 15 μg of total RNA serving as template. Cyanine-labeled Cy3 or Cy5-dUTP (Amersham Bioscience) and pdN6 random hexamers (GE healthcare) were used in reverse transcription reactions utilizing SuperScript II (Invitrogen, CA) for cDNA synthesis. Fluorescent genomic DNA was generated as described in (Girgis et al., 2007). Genomic DNA served as a universal reference for all hybridizations. E. coli genomic DNA was fragmented (500 – 2000 bp) using mechanical shearing, and subjected to Cy3 or Cy5-dUTP labeling using the BioPrime DNA labeling system (Invitrogen, CA).
 
Channel 2
Source name evolved temperature up shift 16min
Organism Escherichia coli K-12
Characteristics MG1655
Treatment protocol Physiological perturbations were carried out under a controlled environment in the context of bioreactor (Bioflo 110, New Brunswick Scientific) growth. Thermoelectric sensors and heaters were used to shift temperature profiles between 250 C and 370 C, and polarographic dissolved oxygen sensors (Mettler Toledo) and nitrogen gas was used to rapidly change oxygen saturation between anaerobic (0% dissolved oxygen) and aerobic (16-21% dissolved oxygen) condition.
Growth protocol Bacteria were grown in batch or bioreactor vessels in M9 minimal media supplemented with 0.4% glucose.
Extracted molecule total RNA
Extraction protocol RNA extraction: A hot-phenol procedure was used to extract total RNA. Cell pellets were lysed with 500 μl TE (pH8.0), 50 μl 10% SDS and lysozyme (0.5 mg/ml). Total RNA was extracted sequentially with phenol/chloroform (preheated to 640 C), followed by chloroform/isoamyl alcohol. RNA was precipitated with 1/10 volume of 3M NaOAc (pH 5.2) and 2 volumes of ethanol. After incubating overnight at –200 C, samples were spun down and pellets were washed with ice cold 70% ethanol (prepared with DEPC- H2O). RNA was resuspended in water, DNase treated (RQ1 RNase-free DNase/ Promega, WI) and purified using an RNeasy purification kit (Qiagen, CA).
Label Cy3
Label protocol Fluorescent cDNA was synthesized from total RNA with 15 μg of total RNA serving as template. Cyanine-labeled Cy3 or Cy5-dUTP (Amersham Bioscience) and pdN6 random hexamers (GE healthcare) were used in reverse transcription reactions utilizing SuperScript II (Invitrogen, CA) for cDNA synthesis. Fluorescent genomic DNA was generated as described in (Girgis et al., 2007). Genomic DNA served as a universal reference for all hybridizations. E. coli genomic DNA was fragmented (500 – 2000 bp) using mechanical shearing, and subjected to Cy3 or Cy5-dUTP labeling using the BioPrime DNA labeling system (Invitrogen, CA).
 
 
Hybridization protocol Cy-dye labeled genomic DNA and RNA-derived cDNA probes were combined with a hybridization buffer (5X SSC, 0.1% SDS, 50% formamide, and 10 μg Salmon sperm DNA) and hybridized for ~ 16 hours at 42 C to a DNA microarray.
Scan protocol Microarray slides were scanned on a GenePix 4000B scanner (Axon Instruments), and fluorescence data for each of the duplicates on each slide were analyzed using GENEPIX PRO 4.0 software.
Description Tn5 transposon mutants (Girgis et al. PLoS Genetics 3(9): e154 (2007))
Sample data table reports condensed data from 2 technical replicates.
Data processing Elements with poor spot morphology or exhibiting uneven hybridization caused by dust particles or scratches, were flagged manually and excluded from further analyses. After local background subtraction and global normalization relative to the genomic DNA reference, duplicate measurements on the same array were averaged, yielding a single vector for each time-point across a perturbation. The data from all hybridizations were combined into a matrix and scaled relative to each other using quantile-normalization as implemented in the Matlab Bioinformatics toolbox. Biological duplicates from all experiments were averaged, leading to a single set of time-series data for each perturbation. Seven time-points were assayed for each perturbation, corresponding to 0, 4, 8, 12, 20, 28, and 44 minutes post transition.
 
Submission date Mar 17, 2008
Last update date May 13, 2008
Contact name yirchung Liu
E-mail(s) [email protected]
Organization name Princeton University
Department Molecular Biology
Lab Tavazoie
Street address 220 Carl Icahn Laboratory
City Princeton
State/province NJ
ZIP/Postal code 08544
Country USA
 
Platform ID GPL6570
Series (1)
GSE10855 MG1655 temperature O2 response normalized data Tavazoie

Data table header descriptions
ID_REF
VALUE log2 of PRE_VALUE
PRE_VALUE quantile-normalized RNA/DNA ratio

Data table
ID_REF VALUE PRE_VALUE
1 10.8037 1787.42
2 10.6129 1566.02
3 10.5975 1549.42
4 8.2280 299.83
5 8.6557 403.30
6 6.4074 84.88
7 11.1635 2293.76
8 8.1743 288.88
9 7.3444 162.51
10 8.4531 350.47
11 7.1516 142.18
12 10.0249 1041.82
14 7.6581 201.99
15 8.4547 350.85
17 7.1423 141.27
18 9.6629 810.61
19 10.0858 1086.76
20 9.0504 530.22
21 9.0953 546.96
24 9.5391 743.99

Total number of rows: 2612

Table truncated, full table size 47 Kbytes.




Supplementary file Size Download File type/resource
GSM275403_12592781_rep1.gpr.gz 454.1 Kb (ftp)(http) GPR
GSM275403_12592781_rep2.gpr.gz 454.5 Kb (ftp)(http) GPR
Processed data included within Sample table

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