|
| Status |
Public on Aug 01, 2019 |
| Title |
Sinorhizobium meliloti RFF231 pMB861, biological rep3 |
| Sample type |
RNA |
| |
|
| Source name |
rpoH1rpoH2 deletion strain RFF231 pMB861 (strain carrying codon optimized Ca. L. asiaticus rpoH on pSRKGm)
|
| Organism |
Sinorhizobium meliloti |
| Characteristics |
genotype/variation: rpoH1rpoH2 deletion
strain: RFF231 pMB861
|
| 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 |
Starter cultures were diluted to ~0.1 OD-600 in 20 ml M9 sucrose medium with 50 ug ml-1 Gm and 500 ug ml-1 Sm, in 250 ml baffled flasks, and allowed to grow to mid-exponential phase (~0.5 to ~0.7 OD-600), with replicates from a single experiment grown to within ~0.1 OD-600 units of each other, and shaking (250 rpm) at 30°C. At mid-exponential phase, IPTG was added to a final concentration of 0.5 mM and the cells were grown for one hour before harvest.
|
| 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 |
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
|
| Scan protocol |
Chips were scanned at the Stanford PAN facility using an Affymetrix Scanner 3000 7G
|
| Data processing |
The data were analyzed using Partek Genomics Suite 6.6 software, with the following settings: RMA for background correction; quantile normalization; log base 2 transformation; median polish summarization
|
| |
|
| Submission date |
Jan 11, 2019 |
| Last update date |
Aug 01, 2019 |
| 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 (2) |
| GSE124982 |
Sinorhizobium meliloti as a model system to study Ca. Liberibacter asiaticus regulators. [RpoH] |
| GSE124984 |
Sinorhizobium meliloti as a model system to study Ca. Liberibacter asiaticus regulators. |
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