|
| Status |
Public on Mar 26, 2009 |
| Title |
Threonine addition |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
E. coli cells 0 min before perturbation
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
stress: control (before perturbation)
|
| Treatment protocol |
Amino acid or nucleotide added to final concentration of 1 mM not exceeding 1/100th volume of culture. After 10 mins, samples were spun down and snap frozen in liquid nitrogen and stored at -80 C
|
| Growth protocol |
E. coli cells grown to mid-exponential phase after 6-8 generations after stationary phase in M9 minimal medium + 0.4 % glucose.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Qiagen RNEASY kit according to directions
|
| Label |
Cy3, Cy5
|
| Label protocol |
10 µg of total RNA were primed with 1 µl of Random hexamers at 70°C for 10 min, then reversed transcribed at 42°C for 2 h in the presence of 200 U SuperScript II RTase (Invitrogen), according to Khodursky et al (PMID 12710666)
|
| |
|
| Channel 2 |
| Source name |
E. coli cells 10 min after specific perturbation
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
stress: Threonine addition
|
| Treatment protocol |
Amino acid or nucleotide added to final concentration of 1 mM not exceeding 1/100th volume of culture. After 10 mins, samples were spun down and snap frozen in liquid nitrogen and stored at -80 C
|
| Growth protocol |
E. coli cells grown to mid-exponential phase after 6-8 generations after stationary phase in M9 minimal medium + 0.4 % glucose.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Qiagen RNEASY kit according to directions
|
| Label |
Cy3, Cy5
|
| Label protocol |
10 µg of total RNA were primed with 1 µl of Random hexamers at 70°C for 10 min, then reversed transcribed at 42°C for 2 h in the presence of 200 U SuperScript II RTase (Invitrogen), according to Khodursky et al (PMID 12710666)
|
| |
|
| |
| Hybridization protocol |
Samples were loaded onto cDNA microarrays and hybridized at 65 C for 5-7 hours. according to Khodursky et al (PMID 12710666)
|
| Scan protocol |
Scanned on GenePix4000B scanner.
|
| Description |
2-3 biological replicates. Comparison of total RNA abundance 10 min after and before addition of Threonine
|
| Data processing |
Images were quantified using GenePix Pro software
Median of intensity data was extracted, LOESS normalized and an ANOVA model was fit using the MAANOVA package in R. The Log (Cy5/Cy3) values were extracted from the VG matrix of the model fit.
|
| |
|
| Submission date |
Mar 26, 2009 |
| Last update date |
Mar 26, 2009 |
| Contact name |
Dipen Sangurdekar |
| E-mail(s) |
dps@genomics.princeton.edu
|
| Organization name |
Princeton University
|
| Department |
Lewis-Sigler Institute for Integrative Genomics
|
| Street address |
132 Carl Icahn laboratory, Princeton University
|
| City |
Princeton |
| State/province |
NJ |
| ZIP/Postal code |
08544 |
| Country |
USA |
| |
|
| Platform ID |
GPL3503 |
| Series (1) |
| GSE15409 |
Amino acid/Nucleotide perturbation in Escherichia coli |
|
