|
| Status |
Public on Mar 01, 2013 |
| Title |
Bmel-glucose-1 |
| Sample type |
RNA |
| |
|
| Source name |
Brucella melitensis strain 16M (ATCC23456) in 1% glucose
|
| Organism |
Brucella melitensis bv. 1 str. 16M |
| Characteristics |
carbon source: 1% Glucose
|
| Growth protocol |
Brucella melitensis strain 16M (ATCC23456) was grown in brucella broth at 37C for 48hrs to stationary phase. 300ul of culture was added to 30ml of yeast-extract minimal medium (YEMM) containing either 1% glucose or 1% erythritol as the sole carbon source. (Plommet, Zentralbl Bakteriol 275(4):436-50)
B. melitensis 16M cultures were grown in YEMM containing either 1% glucose or 1% erythritol as the sole carbon source to late log phase (OD ~1.0) and prepped for RNA.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the Masterpure RNA Extraction Kit (Epicentre) and DNAse treated with the DNAse Turbo Kit (Ambion). Loss of genomic DNA was confirmed by PCR. cDNA was generated using 10ug RNA and Superscript II RT (Invitrogen).
|
| Label |
Cy3
|
| Label protocol |
Biotin labeling of cDNA products was accomplished using biotin-6-dd-ATP and terminal transferase (Roche).
|
| |
|
| Hybridization protocol |
Hybridization was performed at 42C for 16hr in 300ul of buffer containing 50mM MES, 0.5M NaCl, 10mM EDTA, 0.05% Tween 20, 1nM CPK6 Oligo, 0.2 mg of BSA, and 0.04 mg herring sperm DNA. Chips were washed 3x at room temperature with non-stringent buffer (0.18mM NaCl, 10mM NaH2PO4, 1mM EDTA, pH7.7) and 4x at 42C with stringent wash buffer (100mM MES, 0.1M NaCl, 0.01% Tween 20). Chips were stained with a streptavidin-Cy3 conjugate (Amersham) for 25 min. at room temperature, then washed with wash buffer (Nimblegen).
|
| Scan protocol |
Scanning was performed at a 5um resolution on an Axon 4000b scanner (Axon) and image files were processed using Nimblescan software (Nimblegen)
|
| Description |
This sample is of B. melitensis grown in the presence of 1% glucose (control sample), replicate 1
NC_003317 & NC_003318
|
| Data processing |
The raw data (.pair file) was analyzed with Nimblescan software (Nimblegen) and Robust Multi-Array Analysis (RMA) was used to convert the raw data to .calls files.
RMA-normalized values were used to generate gene-specific average intensities. In cases in which a single copy of a probe was present, that probe value was excluded. Gene-specific valuse for each chip were used to compare the erythritol values to the glucose values using gamma-gamma and log-normal-normal analysis.
|
| |
|
| Submission date |
May 07, 2012 |
| Last update date |
Mar 01, 2013 |
| Contact name |
Erik Petersen |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Wisconsin-Madison
|
| Department |
Pathobiological Sciences
|
| Lab |
Gary Splitter
|
| Street address |
1656 Linden Dr.
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
| |
|
| Platform ID |
GPL15533 |
| Series (1) |
| GSE37819 |
Expression response of Brucella melitensis strain 16M (ATCC23456) to erythritol |
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