|
| Status |
Public on Feb 20, 2013 |
| Title |
DSM10140 cells exposed to 1.25mM H2O2 after 5 min, biological rep2 |
| Sample type |
RNA |
| |
|
| Source name |
DSM10140 cells exposed to 1.25mM H2O2 after 5 min
|
| Organism |
Bifidobacterium animalis subsp. lactis DSM 10140 |
| Characteristics |
strain: DSM10140
|
| Treatment protocol |
Cell treatments for transcriptome studies. Cells were grown to late loge phase at 37oC under anaerobic conditions with pH control at 6.5 after which four 5 ml samples were aseptically harvested into sterile 200 ml centrifuge bottles and immediately suspended in 50 mL pre-warmed 50 mL pre-warmed MP5 media containing 1.25mM H2O2. Cells were harvested for RNA isolation after 5 min (T1) and 20 min (T2). Cells were immediatly suspended in 100 mL of RNAprotect reagent (QIAGEN, Inc., Valencia,, CA) and incubated at room temperature for 10 min then collected by cetrifugation. Control cells were immediatly suspended in 10 mL of RNAprotect and incubated at room temperature for 10 min hen collected by cetrifugation (C1). Cell pellets were stored at -80oC until RNA isolation.
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| Growth protocol |
Bacterial strain and growth conditions. B. animalis ssp. lactis Bl-04 and B. animalis ssp. lactis DSM10140 strains were maintained in a laboratory collection as a glycerol stock at -80oC and propagated anaeirobically at 37oC in Mp5 broth. Working cultures were prepared from stock cultures through two successive transfers (0.1% inocula) in MP5 broth at 37oC for 18 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation. Cell pellets were thawed at room temperature, then carefully suspended in 1 ml of lysozyme solution (20 mg/ml in TE buffer) that also contained 20 Units of mutanolysin (Sigma, St. Louis, MO). This mixture was incubated at 37oC for 30 minutes in a shaker incubator at 240 rpm, then total RNA was isolated from each of the cell samples using the Aurum Total RNA Mini Kit (Bio-Rad Laboratories, Hercules, CA) following procedures recommended by the vendor.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cDNA were prepared according to the standard Affymetrix protocol from 10 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
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| |
|
| Hybridization protocol |
Following fragmentation, 10 ug of cDNA were hybridized for 16 hr at 55oC on a custom Affymetrix Bifidobacteria Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400 according to the Affymetrix protocol.
|
| Scan protocol |
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A according to the Affymetrix protocol..
|
| Description |
Gene expression data from DSM10140 cells exposed to 1.25mM H2O2 after 5 min, biological rep2
|
| Data processing |
The data were analyzed with Bioconductor in the open statistical platform R version 2.9.0 using the robust multiarray average (RMA) muti-species method.
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| |
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| Submission date |
Feb 19, 2013 |
| Last update date |
Feb 20, 2013 |
| Contact name |
Taylor S Oberg |
| E-mail(s) |
taylor.oberg@aggiemail.usu.edu
|
| Organization name |
Utah State University
|
| Department |
NDFS
|
| Lab |
Broadbent
|
| Street address |
8700 Old Main
|
| City |
Logan |
| State/province |
UT |
| ZIP/Postal code |
84322 |
| Country |
USA |
| |
|
| Platform ID |
GPL16692 |
| Series (1) |
| GSE44382 |
Expression data from Bifidobacterium animalis ssp. lactis strains exposed to hydrogen peroxide stress |
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