|
| Status |
Public on May 13, 2008 |
| Title |
temperature_Up_28min |
| 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 |
temperature up shift, 28min
|
| 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 |
E. coli strain MG1655
Sample data table reports condensed data from 2 biological replicates (2 technical_replicates/biological_replicate).
CH1: Cy5-rep1, Cy5-rep2; CH2: Cy3-rep1, Cy3-rep2
|
| 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.
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| |
|
| 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 |
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