|
| Status |
Public on Dec 01, 2015 |
| Title |
∆fur ∆ryhB Aerobic B |
| Sample type |
RNA |
| |
|
| Source name |
Fur-minus_RyhB-minus_cDNA_Aerobic
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
genotype/variation: lacking the transcription factor Fur and the small RNA RyhB
culture condition: Aerobic cultures
|
| Treatment protocol |
Cells were treated with a stop solution of phenol and ethanol (Khodursky et al, Methods in Molecular Biology 2003), spun down, and flash frozen and stored at -80°C.
|
| Growth protocol |
Cells were grown aerobically (70% N2, 25% O2, and 5% CO2) or anaerobically (95% N2 and 5% CO2) until mid-log phase (OD600 of ~0.3-0.35) in MOPS minimal glucose media containing 10 µM FeSO4.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using a hot phenol method (Khodursky et al, Methods in Molecular Biology 2003).
|
| Label |
Cy3 monoreactive dye
|
| Label protocol |
10 µg of RNA was reverse transcribed with amino-allyl dUTP and actinomycin D (Cho et. al, Nature Biotechnology 2009). Purified 1st-strand, amino-allyl labeled cDNA was coupled to Amersham Cy3 monoreactive dye (GE Healthcare) in 0.1 M sodium carbonate buffer for 1 hour, at room temperature, in the dark. The reaction was stopped with NH2OH. The purified, labeled cDNAs were fragmented with DNase I (0.1 U per ug of cDNA) for 10 min at 37°C. DNase I was then inactivated at 95°C for 10 min; samples from RyhB- and Fur-RyhB- strains also included 0.85 mM EDTA in this reaction. Cy3-labelled cDNAs were precipitated and fragmentation was verified by running a 2% agarose gel.
|
| |
|
| Hybridization protocol |
~0.6-1.5 ug of Cy3-labelled cDNA was hybridized to a custom-made E. coli K-12 MG1655 tiled genome microarray. Hybridization was performed overnight for ~17 hours at 42°C in a NimbleGen Hybridization System 4, according to the NimbleGen hybridization protocol.
|
| Scan protocol |
Hybridized tiling arrays were scanned using a GenePix 4000B microarray scannner (Axon Instruments). The PMT for 532 nm was adjusted so that the median fluorescence was just below 100.
|
| Data processing |
Raw probe intensities were normalized across all samples using the Robust Multichip Average (RMA) algorithm in the NimbleScan software package (version 2.5).
RMA normalized probe data was uploaded into the Mochiview visualization software where data cooresponding to the plus and minus strands were subtracted to get a strand-specific probe intesenity per base as reported in the processed data files.
|
| |
|
| Submission date |
Nov 12, 2015 |
| Last update date |
Dec 01, 2015 |
| Contact name |
Patricia J Kiley |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Wisconsin - Madison
|
| Department |
Biomolecular Chemistry
|
| Street address |
420 Henry Mall, Room 1135 Biochemistry Building
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
| |
|
| Platform ID |
GPL8708 |
| Series (2) |
| GSE74930 |
The impact of anaerobiosis on expression of the iron-responsive Fur and RyhB Regulons [expression microarray] |
| GSE74933 |
The impact of anaerobiosis on expression of the iron-responsive Fur and RyhB Regulons |
|
