|
| Status |
Public on Dec 05, 2014 |
| Title |
YPIIIanaerobvsaerob_log_GEO_sample02 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
WT_aerobic_exponential phase
|
| Organism |
Yersinia pseudotuberculosis YPIII |
| Characteristics |
strain: YPIII pIB1 wild type
growth condition: aerobic
growth phase: exponential phase
|
| Growth protocol |
Y. pseudotuberculosis wild-type strain YPIII was grown at 25°C in LB medium supplemented with 10 g/l glucose and 0.2 M HEPES buffer under aeration or under an-aerobic growth conditions (in a nitrogen atmosphere) to exponential phase (OD600=0.5). Addition of the HEPES buffer was required to avoid acidification of the medium below pH 5.0 due to mixed acid fermentation, resulting in a significant growth reduction/stop. Additional glucose was added to maximize energy production and growth rate under aerobic growth condi-tions. Absence of oxygen in the culture medium was tested by gas chromatography (GC-WLD, Carlo Erba Vega Series 6000) coupled with a detector coupled with an integrator (Spectra-Physics, SP4270) using a Poropak QS (100-120 Mesh) column and helium (Westalen 4.6) at 300 kPa.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was performed using the SV Total RNA Isolation System (Promega)
|
| Label |
Cy5
|
| Label protocol |
RNA samples were labelled either with Cy3 (anaerob) or Cy5 (aerob) using the ULS fluorescent labelling kit (Kreatech, Germany)
|
| |
|
| Channel 2 |
| Source name |
WT_anaerobic_exponential phase
|
| Organism |
Yersinia pseudotuberculosis YPIII |
| Characteristics |
strain: YPIII pIB1 wild type
growth condition: anaerobic
growth phase: exponential phase
|
| Growth protocol |
Y. pseudotuberculosis wild-type strain YPIII was grown at 25°C in LB medium supplemented with 10 g/l glucose and 0.2 M HEPES buffer under aeration or under an-aerobic growth conditions (in a nitrogen atmosphere) to exponential phase (OD600=0.5). Addition of the HEPES buffer was required to avoid acidification of the medium below pH 5.0 due to mixed acid fermentation, resulting in a significant growth reduction/stop. Additional glucose was added to maximize energy production and growth rate under aerobic growth condi-tions. Absence of oxygen in the culture medium was tested by gas chromatography (GC-WLD, Carlo Erba Vega Series 6000) coupled with a detector coupled with an integrator (Spectra-Physics, SP4270) using a Poropak QS (100-120 Mesh) column and helium (Westalen 4.6) at 300 kPa.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was performed using the SV Total RNA Isolation System (Promega)
|
| Label |
Cy3
|
| Label protocol |
RNA samples were labelled either with Cy3 (anaerob) or Cy5 (aerob) using the ULS fluorescent labelling kit (Kreatech, Germany)
|
| |
|
| |
| Hybridization protocol |
300ng of each Cy3- and Cy5-labelled RNA and hybridized to the microarray at 65°C for 17 hours using the Agilent Hybridization Chamber according to the Agilent instructions
|
| Scan protocol |
The array were scanned at a resolution of 5μm using the Axon GenePix Personal 4100A microarray scanner (Axon instruments) and features were extracted using GenePix Pro 6
|
| Description |
Biological replicate 2 of 4.
|
| Data processing |
Microarray data processing was done using the Limma package (Smyth, 2005) from the R/Bioconductor framework (Gentleman et al., 2004). Unprocessed array intensity values were read-in using function read.maimages and overall array and hybridization qualities were assessed by evaluation of control probe intensities and exploratory data analysis. Quality assessed/controlled intensities were subsequently normalized using the variance stabilization normalization method (Huber et al., 2002) with background substraction as implemented in the vsn2 function of the Bioconductor package vsn.
|
| |
|
| Submission date |
Apr 02, 2014 |
| Last update date |
Dec 06, 2014 |
| Contact name |
Kemal Avican |
| E-mail(s) |
kemal.avican@umu.se
|
| Organization name |
Umea University
|
| Department |
Department of Molecular Biology
|
| Lab |
Maria Fällman's Lab
|
| Street address |
Building 6L
|
| City |
Umea |
| ZIP/Postal code |
90187 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL15095 |
| Series (2) |
| GSE56475 |
Complex in vivo RNA-seq analysis reveal reprogramming of Yersinia from virulent to persistent mode during infection [microarray I] |
| GSE56477 |
Complex in vivo RNA-seq analysis reveal reprogramming of Yersinia from virulent to persistent mode during infection |
|
