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| Status |
Public on Dec 31, 2014 |
| Title |
H02: A_T4 vs A_T5 |
| Sample type |
RNA |
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| Channel 1 |
| Source name |
T4 sample in experiment A
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| Organism |
Clostridium perfringens SM101 |
| Characteristics |
strain: SM101
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| Growth protocol |
An exponential-phase inoculum was prepared by mixing 1 ml of the overnight culture and 9 ml FTM followed by 4 h incubation; the fast dividing vegetative cells produced substantial amounts of gas. An inoculum (1:10,000, v:v) was added to 500 ml of pre-reduced mDS medium and incubated at 37°C. This sporulating culture was continuously mixed using a magnetic stirrer. Samples of the sporulating culture were taken every 30 min starting 2 h after inoculation. A 3 ml mixture of acidified phenol-ethanol (v:v=1:9) was added to 12 ml culture samples immediately upon sampling. After short mixing using a vortex, this sample was kept on ice for 30 min. Cells/spores were then harvested by centrifugation at 16,000 ×g for 5 min at 4°C, and resuspended in ice cold TRIzol Reagent (Ambion, US).
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| Extracted molecule |
total RNA |
| Extraction protocol |
0.6 ml aliquots of the mixture were transferred to Lysing Matrix B tubes prefilled with 0.1mm silica spheres. These samples were frozen in liquid N2 and kept at -80°C until use. For final RNA extraction, 0.4 ml ice-cold TRIzol Reagent was added to the frozen sample and homogenization was performed immediately without thawing using Savant FastPrep FP120 (Qbiogene, Carlsbad, US) for 40 sec at Speed 6, in total 3 times with interval cooling on ice. After phase separation, RNA precipitation and washing was performed as described in the instructions of the manufacturer. The total RNA extracts were further purified using a RNeasy kit (QIAGEN, Hilgen, Germany). On-column DNA digestion was performed during purification using RNase-Free DNase (QIAGEN, Hilgen, Germany).
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| Label |
Cy3
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| Label protocol |
3 µg of total RNA was subjected to cDNA synthesis using random nonamer priming and indirect labeling approaches following the manufacture instruction of the Cyscribe Postlabelling kit (Amersham Biosciences). For each sample at each time point, both Cy3- and Cy5-labeled cDNA was prepared.
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| Channel 2 |
| Source name |
T5 sample in experiment A
|
| Organism |
Clostridium perfringens SM101 |
| Characteristics |
strain: SM101
|
| Growth protocol |
An exponential-phase inoculum was prepared by mixing 1 ml of the overnight culture and 9 ml FTM followed by 4 h incubation; the fast dividing vegetative cells produced substantial amounts of gas. An inoculum (1:10,000, v:v) was added to 500 ml of pre-reduced mDS medium and incubated at 37°C. This sporulating culture was continuously mixed using a magnetic stirrer. Samples of the sporulating culture were taken every 30 min starting 2 h after inoculation. A 3 ml mixture of acidified phenol-ethanol (v:v=1:9) was added to 12 ml culture samples immediately upon sampling. After short mixing using a vortex, this sample was kept on ice for 30 min. Cells/spores were then harvested by centrifugation at 16,000 ×g for 5 min at 4°C, and resuspended in ice cold TRIzol Reagent (Ambion, US).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
0.6 ml aliquots of the mixture were transferred to Lysing Matrix B tubes prefilled with 0.1mm silica spheres. These samples were frozen in liquid N2 and kept at -80°C until use. For final RNA extraction, 0.4 ml ice-cold TRIzol Reagent was added to the frozen sample and homogenization was performed immediately without thawing using Savant FastPrep FP120 (Qbiogene, Carlsbad, US) for 40 sec at Speed 6, in total 3 times with interval cooling on ice. After phase separation, RNA precipitation and washing was performed as described in the instructions of the manufacturer. The total RNA extracts were further purified using a RNeasy kit (QIAGEN, Hilgen, Germany). On-column DNA digestion was performed during purification using RNase-Free DNase (QIAGEN, Hilgen, Germany).
|
| Label |
Cy5
|
| Label protocol |
3 µg of total RNA was subjected to cDNA synthesis using random nonamer priming and indirect labeling approaches following the manufacture instruction of the Cyscribe Postlabelling kit (Amersham Biosciences). For each sample at each time point, both Cy3- and Cy5-labeled cDNA was prepared.
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| Hybridization protocol |
Labeled cDNA mixtures were subsequently concentrated in a Hetovac VR-1 (Heto Lab Equipment A/S, Birkerod, Denmark), incubated at 98°C for 3 min, and cooled at room temperature for 5 min. After the addition of 2× GEX HI-RPM hybridization buffer (Agilent Technologies, Palo Alto, CA), each mixture was applied to an Agilent 4×44K array (Agilent Technologies, Palo Alto, CA).
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| Scan protocol |
Slides were scanned with a ScanArray Express 4000 scanner (Perkin Elmer, Wellesley, MA), image analysis and processing were performed using the ImaGene Version 7.5 software (BioDiscovery Inc., Marina Del Rey, CA, USA). The microarrays were scanned at different intensities. For each of the individual microarrays the best scan was selected on the basis of signal distribution (combination of a low number of saturated spots and a low number of low signal spots).
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| Description |
Raw data file (Cy3): SlideA_Cy3
Raw data file (Cy5): SlideA_Cy5
Meta region on slide: 3
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| Data processing |
The data were normalized using Lowess normalization as available in MicroPrep. The data were corrected for inter-slide differences on the basis of total signal intensity per slide using Postprep. The median intensity of the different probes per gene was selected as the gene expression intensity. CyberT was used to compare the different transcriptomes, taking into account the replicates of each of the conditions. This analysis resulted in a gene expression ratio and false discovery rate (FDR) for each gene. Genes with FDR values <0.05 were considered to be statistically significant.
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| Submission date |
Jul 21, 2014 |
| Last update date |
Dec 31, 2014 |
| Contact name |
Yinghua Xiao |
| E-mail(s) |
yinghua.xiao@wur.nl
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| Organization name |
Wageningen University
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| Street address |
Bornse Weilanden 9
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| City |
Wageningen |
| ZIP/Postal code |
6700AA |
| Country |
Netherlands |
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| Platform ID |
GPL18980 |
| Series (1) |
| GSE59616 |
Genome-wide transcriptional profiling of Clostridium perfringens SM101 during sporulation |
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| Supplementary data files not provided |
| Processed data are available on Series record |
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