|
| Status |
Public on Oct 02, 2012 |
| Title |
Sinorhizobium meliloti AB3, heat shock, biological rep3 |
| Sample type |
RNA |
| |
|
| Source name |
rpoH2 insertion mutant
|
| Organism |
Sinorhizobium meliloti |
| Characteristics |
strain: AB3 (Valerie Oke)
genotype/variation: rpoH2 insertion mutant
treatment: 42°C for 15 minutes in LB/MC medium
|
| Treatment protocol |
Stop solution (5% buffer equilibrated phenol in ethanol) was added to bacterial cultures, at one tenth the culture volume, before centrifugation at 4 degrees celsius. Bacterial cell pellets were frozen in liquid nitrogen and stored at -80 degrees celsius until RNA extraction.
|
| Growth protocol |
For the heat shock experiment: cultures were diluted to OD595=0.05 in 65 ml LB/MC and allowed to grow to mid exponential phase (OD595=0.5-0.7). Each wild-type culture was split such that 30 ml remained at 30°C for 15 minutes as a control, and 30 ml was heat-shocked for 15 minutes at 42°C.
For the stationary phase experiment: overnight cultures were grown in LB/MC medium, diluted to OD595 = 0.05 the next day, and allowed to grow overnight. Cells (8 ml) were washed twice and diluted to OD595=0.05 in 300 ml M9 sucrose minimal medium. Cultures were incubated with shaking for 48 hours, until late stationary phase.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using a Qiagen RNeasy kit with modifications as described by Barnett et al. 2004 Proc Natl Acad Sci USA volume 101 pages 16636 to 16641.
|
| Label |
biotin
|
| Label protocol |
cDNA was fragmented and biotinylated as described by Barnett et al. 2004.
|
| |
|
| Hybridization protocol |
For the heat shock experiment, four micrograms of biotinylated cDNA was hybridized to each chip at 48 degrees celsius for 16 hours as described by Barnett et al. 2004. GeneChips were washed and stained in the Affymetrix Fluidics Station 400. The stationary phase experiment was performed similarly, except twelve micrograms of biotinylated cDNA was hybridized to each chip.
|
| Scan protocol |
Chips were scanned at the Stanford PAN facility using an Affymetrix scanner 3000 7G
|
| Description |
rpoH2-HS-3
|
| Data processing |
The data were analyzed using Affymetrix software GCOS v1.4 with Affymetrix default analysis setting and global scaling as the normalization method. The mean target intensity of each array was arbitrarily set to 500. Affymetrix Data Mining Tool v3.1 was used for data mining as previously described by Barnett et al. 2004.
|
| |
|
| Submission date |
Mar 01, 2012 |
| Last update date |
Oct 02, 2012 |
| Contact name |
Melanie J Barnett |
| Organization name |
Stanford University
|
| Department |
Biology
|
| Lab |
Sharon R. Long
|
| Street address |
371 Jane Stanford Way
|
| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platform ID |
GPL9757 |
| Series (1) |
| GSE36186 |
Dual RpoH sigma factors in Sinorhizobium meliloti |
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