|
| Status |
Public on Sep 18, 2013 |
| Title |
WT_CTRL_H2O2_REPLICATE_1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
WT_CTRL_REP1
|
| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S |
| Characteristics |
genotype/variation: WT
stress: CTRL
|
| Extracted molecule |
total RNA |
| Extraction protocol |
High Pure RNA isolation kit (Roche)
|
| Label |
Cy3
|
| Label protocol |
Incorporation of dye using about 1200U Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA), 40ng/ul random hexamer primers, 4nmol Cy dUTP (GE Healthcare, Fairfield, CT), and 80U RNAse Inhibitor (Roche, Indianapolis, IN), in 60ul reactions. Reverse transcription was performed for 2h at 42˚C, and the pH adjusted with 50mM NaOH. After a 10min incubation at 70˚C reactions were neutralized using HCl. Labeled cDNA was purified using the QIAGEN PCR purification kit as suggested by the manufacturer (Qiagen, Valencia, CA).
|
| |
|
| Channel 2 |
| Source name |
WT_H2O2_REP1
|
| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S |
| Characteristics |
genotype/variation: WT
stress: H2O2
growth protocol: wt salmonella grown to OD600 0.4 in 25 ml LB medium, and treated with 1.5 mM H2O2 for 20 min. replicate 01
|
| Extracted molecule |
total RNA |
| Extraction protocol |
High Pure RNA isolation kit (Roche)
|
| Label |
Cy5
|
| Label protocol |
Incorporation of dye using about 1200U Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA), 40ng/ul random hexamer primers, 4nmol Cy dUTP (GE Healthcare, Fairfield, CT), and 80U RNAse Inhibitor (Roche, Indianapolis, IN), in 60ul reactions. Reverse transcription was performed for 2h at 42˚C, and the pH adjusted with 50mM NaOH. After a 10min incubation at 70˚C reactions were neutralized using HCl. Labeled cDNA was purified using the QIAGEN PCR purification kit as suggested by the manufacturer (Qiagen, Valencia, CA).
|
| |
|
| |
| Hybridization protocol |
42C overnight in standard hybridization buffer (Roche Nimblegen)
|
| Scan protocol |
GenePix4000B scanner (Molecular Devices, Sunnyvale, CA) using Acuity 4.0 software.
|
| Description |
EM_420454_WC1_Cy3_ch1_WP1_Cy5_ch2
|
| Data processing |
Signal extraction and quantitation with Roche’s NimbleScan 2.4 package
log base 10 ratios of contributions to total signal (experimental condition / control)
normalized based on total signal for each channel
|
| |
|
| Submission date |
Dec 04, 2011 |
| Last update date |
Sep 18, 2013 |
| Contact name |
Michael McClelland |
| E-mail(s) |
mcclelland.michael@gmail.com
|
| Phone |
858-336-9554
|
| Organization name |
University of California, Irvine
|
| Department |
Microbiology & Molecular Genetics
|
| Street address |
132 Med Surge I
|
| City |
Irvine |
| State/province |
CA |
| ZIP/Postal code |
92697 |
| Country |
USA |
| |
|
| Platform ID |
GPL14855 |
| Series (1) |
| GSE34134 |
Role of the ArcAB two-component system in oxidative stress |
|
