|
| Status |
Public on Jan 01, 2015 |
| Title |
Oxidative stressed replicate 1, dye swap |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
100 mM hydrogen peroxide-stressed cells, 10 minutes
|
| Organism |
Methylorubrum extorquens AM1 |
| Characteristics |
strain: AM1
genotype: Wild type
|
| Treatment protocol |
40 ml samples were removed, transferred to 250 ml flasks, and exposed to appropriate stressor; duplicate control samples were removed and left untreated. Samples were placed in shaker at 28C for 10 minutes.
|
| Growth protocol |
Cells were grown on minimal salts medium containing 25 mM methanol in a chemostat (BioFlo 110 Modular Fermentor, New Brunswick Scientific, Edison, NJ) under steady-state conditions at 28C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were placed into 1/10 volume ice-cold "Stop Solution" (5% v/v water saturated phenol at pH 7.0, 95% v/v absolute ethanol). Cells were extracted via centrifugation at 4C for 8 min at 4500 x g. RNA was extracted using Qiagen's RNeasy Mini Kit (yeast protocol). Cells were lysed with a Mini-beadbeater 8 (BioSpec Products, Bartlesville, OK) at 4C with 600 uL 0.1-mm zirconium-silica beads (BioSpec Products).
|
| Label |
Alexa Fluor 647
|
| Label protocol |
5 ug DNase-I treated total RNA and 4 ug random decamers were used for cDNA synthesis incorporating amino allyl dUTP as per Amino Allyl cDNA Labelling Kit (Ambion, Austin, TX). Following overnight ethanol precipitation at -80C and 45 min microcentrifugation at 20,800 x g at 4C, cDNA was coupled to either Alexa Fluor 555 ot Alexa Fluor 647 reactive dye dissolved in 3 uL high quality DMSO.
|
| |
|
| Channel 2 |
| Source name |
Unstressed cells, 10 minutes
|
| Organism |
Methylorubrum extorquens AM1 |
| Characteristics |
strain: AM1
genotype: Wild type
|
| Treatment protocol |
40 ml samples were removed, transferred to 250 ml flasks, and exposed to appropriate stressor; duplicate control samples were removed and left untreated. Samples were placed in shaker at 28C for 10 minutes.
|
| Growth protocol |
Cells were grown on minimal salts medium containing 25 mM methanol in a chemostat (BioFlo 110 Modular Fermentor, New Brunswick Scientific, Edison, NJ) under steady-state conditions at 28C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were placed into 1/10 volume ice-cold "Stop Solution" (5% v/v water saturated phenol at pH 7.0, 95% v/v absolute ethanol). Cells were extracted via centrifugation at 4C for 8 min at 4500 x g. RNA was extracted using Qiagen's RNeasy Mini Kit (yeast protocol). Cells were lysed with a Mini-beadbeater 8 (BioSpec Products, Bartlesville, OK) at 4C with 600 uL 0.1-mm zirconium-silica beads (BioSpec Products).
|
| Label |
Alexa Fluor 555
|
| Label protocol |
5 ug DNase-I treated total RNA and 4 ug random decamers were used for cDNA synthesis incorporating amino allyl dUTP as per Amino Allyl cDNA Labelling Kit (Ambion, Austin, TX). Following overnight ethanol precipitation at -80C and 45 min microcentrifugation at 20,800 x g at 4C, cDNA was coupled to either Alexa Fluor 555 ot Alexa Fluor 647 reactive dye dissolved in 3 uL high quality DMSO.
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed as per the Agilent 60-mer processing protocol (Sure-Hyb chamber/SSPE wash with the Agilent Stabilization and Drying Solution) using a microhybridization oven (Bellco Biotechnology, Vienland, NJ). 40 pmol Alexa Fluor 555- and Alexa Fluor 647-labeled cDNAs were cohybridized. Following hybridization, slides were washed and dried and stored in nitrogen-purged SmartDesiccator (Terra Universal, Fullerton, CA) until scanning.
|
| Scan protocol |
Slides were sent to Center for Array Technologies at the University of Washington (Seattle, WA) and scanned using an Agilent G2565AA microarray scanner.
|
| Description |
Biological replicate 1 of 3; dye swap
|
| Data processing |
Scanned images were processed by Agilent Feature Extraction software Ver. 7.5.1. using default settings for Agilent arrays, Linear & Lowess method for normalization, and PMT setting of 10, 50, and 100.
|
| |
|
| Submission date |
Apr 10, 2009 |
| Last update date |
Jan 01, 2015 |
| Contact name |
Jonathan A. Miller |
| E-mail(s) |
[email protected]
|
| URL |
https://depts.washington.edu/mllab/
|
| Organization name |
University of Washington
|
| Department |
Microbiology
|
| Lab |
Lidstrom Laboratory
|
| Street address |
616 NE Northlake Place
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195 |
| Country |
USA |
| |
|
| Platform ID |
GPL6262 |
| Series (1) |
| GSE15631 |
Formaldehyde stress response in Methylobacterium extorquens AM1 |
|
