|
| Status |
Public on Apr 02, 2014 |
| Title |
Fur KO Gent 3 |
| Sample type |
RNA |
| |
|
| Source name |
Gent treated ∆fur E coli cells at 60 min
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
genotype: ∆fur
treatment: 5 ug/ml gentamicin
timing: 60 min
|
| Treatment protocol |
Cells were treated with their respective treatments for 1 hour before sample collection for RNA extraction.
|
| Growth protocol |
Cells were grown in 10 mL LB in 250 mL baffled flasks at 37°C and 300 RPM in a light-protected, humidity-controlled incubator shaker to an OD600 of ~0.3 before transfer to 14 mL polypropylene tubes and indicated treatments applied.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was obtained using RNAprotect (Qiagen), the RNeasy Protect Bacteria Mini Kit (Qiagen) and Turbo DNA-free (Life Technologies) DNase treatment according to respective manufacturer instructions.
|
| Label |
biotin
|
| Label protocol |
cDNAs were prepared from 10 µg total RNA through a random primed reverse transcription using SuperScript II (Invitrogen, Carlsbad, CA). The RNAs were digested with the addition of 1M NaOH and incubation at 65ºC for 30 minutes. The mixtures were neutralized with the addition of 1M HCl. The cDNAs were purified using QIAquick MinElute PCR purification columns (Qiagen, Valencia, CA), following the manufacturer’s protocol. The cDNAs (3 – 5 μg) were fragmented to a size range of 50-200 bases with DNase I (0.6 U/ μg cDNA) at 37 ºC for 10 minutes followed by inactivation of the enzyme at 98 ºC for 10 minutes.
|
| |
|
| Hybridization protocol |
Fragmented, biotinylated cDNAs were combined with hybridization cocktail and hybridized to Affymetrix arrays for 16-18 hours at 45 ºC and 60 rpm.
|
| Scan protocol |
Arrays were washed and stained on Affymetrix Fluidics Station 450s using the appropriate Affymetrix protocol. The stained arrays were scanned at 532 nm using an Affymetrix GeneChip Scanner 3000.
|
| Description |
Gent treated ∆fur E coli cells at 60 min
|
| Data processing |
CEL files for the resulting expression profiles were background adjusted and RMA normalized using RMAExpress. Statistical analysis was performed in MATLAB.
|
| |
|
| Submission date |
Mar 24, 2014 |
| Last update date |
Apr 02, 2014 |
| Contact name |
Jason H Yang |
| E-mail(s) |
[email protected]
|
| Organization name |
MIT / Broad Institute
|
| Department |
Biological Engineering
|
| Lab |
James Collins
|
| Street address |
415 Main St, Rm 2017
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL3154 |
| Series (1) |
| GSE56133 |
Antibiotics induce redox-related physiological alterations as part of their lethality |
|
