|
| Status |
Public on Jan 31, 2016 |
| Title |
4H-02_Vivo |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
R20291 extracted after 4h in vivo growth
|
| Organism |
Clostridioides difficile |
| Characteristics |
strain background: R20291
genotype/variation: wild type
|
| Growth protocol |
We did a 9 hours preculture of Clostridium difficile R20291 in Tryptone yeast extract (TY), we centrifuged and resuspended in PBS. Then we gave 5ml of this preparation to 6 mouses per time point (C. difficile monoxenic mouse model). Finally mouse were sacrificed, according European rules, at different time 4, 6, 8, 14 and 38 hours after infection to collect the luminal and mucosa associated bacteria in the caeca.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using RNAPro Blue (FastPrep) and following Qbiogen manufacturer's instructions
|
| Label |
cy3
|
| Label protocol |
10 µg of total RNA and pdN6 primer were heated at 70°C for 5 min, then reversed transcribed at 42°C for 3 h in the presence of 1600 U SuperScript III Reverse Transcriptase (Invitrogen), each dATP, dTTP, dGTP, dCTP. cDNA were then incubated with cy5 or cy3 dyes 1h at room temperature in the dark. DNA samples were labelled using the Bioprime plus array CGH indirect genomic labeling system (invitrogen)
|
| |
|
| Channel 2 |
| Source name |
R20291 grown 14H in PY (reference)
|
| Organism |
Clostridioides difficile |
| Characteristics |
strain background: R20291
genotype/variation: wild type
|
| Growth protocol |
We did a 9 hours preculture of Clostridium difficile R20291 in Tryptone yeast extract (TY), we centrifuged and resuspended in PBS. Then we gave 5ml of this preparation to 6 mouses per time point (C. difficile monoxenic mouse model). Finally mouse were sacrificed, according European rules, at different time 4, 6, 8, 14 and 38 hours after infection to collect the luminal and mucosa associated bacteria in the caeca.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using RNAPro Blue (FastPrep) and following Qbiogen manufacturer's instructions
|
| Label |
cy5
|
| Label protocol |
10 µg of total RNA and pdN6 primer were heated at 70°C for 5 min, then reversed transcribed at 42°C for 3 h in the presence of 1600 U SuperScript III Reverse Transcriptase (Invitrogen), each dATP, dTTP, dGTP, dCTP. cDNA were then incubated with cy5 or cy3 dyes 1h at room temperature in the dark. DNA samples were labelled using the Bioprime plus array CGH indirect genomic labeling system (invitrogen)
|
| |
|
| |
| Hybridization protocol |
Samples were applied to microarrays enclosed in Agilent hybridization chambers for 17h at 65°C. After hybridization, slides were washed with Agilent washing buffers
|
| Scan protocol |
Scanned on an GenePix 4200L dual-channel laser scanner
|
| Description |
Biological replicate 1 of 4.
raw data file: A_4h-02_cy3-420_cy5-390.gpr
|
| Data processing |
Background correction: backgroundCorrect 'normexp', Normalization: normalizeWithinArrays 'loess', Correlation of spot duplicat: duplicateCorrelation, Adjust to linear method: lmFit, Statistical analysis: eBayes, decideTests 'global' 'BY'[statistical results provided as Series Supplementary file].
|
| |
|
| Submission date |
Feb 10, 2012 |
| Last update date |
Jan 31, 2016 |
| Contact name |
Monot Marc |
| E-mail(s) |
mmonot@pasteur.fr
|
| Phone |
+33145688390
|
| Organization name |
Institut Pasteur
|
| Department |
Genomes and Genetics
|
| Lab |
Biomics
|
| Street address |
25, rue du docteur roux
|
| City |
Paris |
| ZIP/Postal code |
75015 |
| Country |
France |
| |
|
| Platform ID |
GPL15218 |
| Series (1) |
| GSE35726 |
Comparison of the expression profiles of R20291 strain and a R20291 strain after 4h, 6h, 8h, 14h, 38h of growth in vivo |
|
