|
| Status |
Public on Jan 12, 2012 |
| Title |
SH1000-targocil-30 min_rep3 (slide 556) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
[EXPT] SH 1000 treated with targocil for30 min
|
| Organism |
Staphylococcus aureus |
| Characteristics |
strain: SH1000
agent: targocil
time: 30 min
|
| Treatment protocol |
For the targocil treatment experiments, S. aureus was grown to an OD600 ~0.4 and challenged with 8x MIC of targocil dissolved in DMSO for 30 min. Simultaneously, an equal amount of DMSO (final concentration 2% (v/v)) was added to the control culture, which was incubated along with the treated cultures. After 30 min, 5 mL aliquot was collected and used as a control for the 30 min treated culture.
|
| Growth protocol |
An overnight S. aureus SH1000 culture was diluted (2% (v/v)) into 20 mL TSB medium in a 50 mL Erlenmeyer flask and grown at 37 °C with shaking at 200 rpm. For the targocil treatment experiments, S. aureus was grown to an OD600 ~0.4 and challenged with 8x MIC of targocil dissolved in DMSO for 30 min. Simultaneously, an equal amount of DMSO (final concentration 2% (v/v)) was added to the control culture, which was incubated along with the treated cultures. After 30 min, 5 mL aliquot was collected and used as a control for the 30 min treated culture.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Bacterial cultures flushed directly into QIAGEN RNA protect before total RNA isolation using QIAGEN RNEasy Midi kit with on column DNAse digestion.
|
| Label |
Cy3
|
| Label protocol |
Labelled with Cy3 and Cy5 during cDNA synthesis from total RNA using Superscript II Kit (Invitrogen) as manufacturers instructions. Samples purified using QIAGEN PCR purification kit before hybridization.
|
| |
|
| Channel 2 |
| Source name |
[CTL] S. aureus SH1000 treated with DMSO for 30 min
|
| Organism |
Staphylococcus aureus |
| Characteristics |
strain: SH1000
agent: control
time: 30 min
|
| Treatment protocol |
For the targocil treatment experiments, S. aureus was grown to an OD600 ~0.4 and challenged with 8x MIC of targocil dissolved in DMSO for 30 min. Simultaneously, an equal amount of DMSO (final concentration 2% (v/v)) was added to the control culture, which was incubated along with the treated cultures. After 30 min, 5 mL aliquot was collected and used as a control for the 30 min treated culture.
|
| Growth protocol |
An overnight S. aureus SH1000 culture was diluted (2% (v/v)) into 20 mL TSB medium in a 50 mL Erlenmeyer flask and grown at 37 °C with shaking at 200 rpm. For the targocil treatment experiments, S. aureus was grown to an OD600 ~0.4 and challenged with 8x MIC of targocil dissolved in DMSO for 30 min. Simultaneously, an equal amount of DMSO (final concentration 2% (v/v)) was added to the control culture, which was incubated along with the treated cultures. After 30 min, 5 mL aliquot was collected and used as a control for the 30 min treated culture.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Bacterial cultures flushed directly into QIAGEN RNA protect before total RNA isolation using QIAGEN RNEasy Midi kit with on column DNAse digestion.
|
| Label |
Cy5
|
| Label protocol |
Labelled with Cy3 and Cy5 during cDNA synthesis from total RNA using Superscript II Kit (Invitrogen) as manufacturers instructions. Samples purified using QIAGEN PCR purification kit before hybridization.
|
| |
|
| |
| Hybridization protocol |
Labelled cDNA hybridised in slides placed in water bath for 16 h at 42 °C. Arrays then washed in 1x SSC (+0.02 % SDS)at room temperature for 2 and 5 min, in 0.1x SSC+0.1%SDS for 5 min, and then in 0.1x SSC for 10 min 2 times prior to drying and scanning.
|
| Scan protocol |
Arrays scanned using an Gene pix Microarray scaner, Gene pix professional, 4200A, axon Instrument scanner and Imagene version Pro 6.0 software.
|
| Description |
Replicate 3
Raw data file: slide 556_CTL_Cy5_EXP_Cy3.txt
Processed data file: slide 556 log2 ratio.txt
An antibiotic that inhibits a late step in wall teichoic acid biosynthesis induces the cell wall stress stimulon in Staphylococcus aureus
|
| Data processing |
Data analysis was carried out using Spotfinder, version 224 , MIDAS2_19, MadRalph_1.0_Final_011503 and SAM. The mean values from each channel were log2 transformed and normalized using the LOWESS algorithm to remove intensity dependent effects within the calculated values. We used MIDAS software for data normalization.
Results of SAM analysis, and merging of replicate hybridizations are provided as supplementary files on the Series record.
In raw data files, channel A = Cy3 dye and channel B = Cy5 dye.
|
| |
|
| Submission date |
Jan 12, 2012 |
| Last update date |
Jan 12, 2012 |
| Contact name |
Arunachalam Muthaiyan, Brian Wilkinson |
| E-mail(s) |
amuthai@gmail.com, bjwilkin@ilstu.edu
|
| Phone |
309-438-7244
|
| URL |
http://www.bio.ilstu.edu/Wilkinson/
|
| Organization name |
Illinois State University
|
| Department |
Biological Sciences
|
| Lab |
Microbiology
|
| Street address |
Julian Hall
|
| City |
Normal |
| State/province |
IL |
| ZIP/Postal code |
61790 |
| Country |
USA |
| |
|
| Platform ID |
GPL3866 |
| Series (1) |
| GSE34441 |
An antibiotic that inhibits a late step in wall teichoic acid biosynthesis induces the cell wall stress stimulon in Staphylococcus aureus |
|
