|
| Status |
Public on Dec 31, 2011 |
| Title |
Genotyping of Isolate CB76 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
CB76
|
| Organism |
Coxiella burnetii |
| Characteristics |
strain: CB76
|
| Growth protocol |
C. burnetii were grown at 35°C on L929 cells using MEM (GIBCO, Invitrogen, Cergy-Pontoise, France) supplemented with 4% SVF (GIBCO) and 1% L-glutamine (GIBCO). Monolayers of cells and the supernatants from three 175 cm² flasks were harvested and incubated with 1% trypsin (GIBCO) for 1 hour at 37°C. Released bacteria were purified from L929 cell debris by differential centrifugation. Purified bacteria were resuspended in 400 µl of PBS and stored at -80°C
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
200 µl of purified bacteria were incubated for 30 min at 70°C with 200 µl of AL lysis buffer (Qiagen, Courtaboeuf, France) and 20 µl of proteinase K (Qiagen). gDNA was extracted and purified using a QiaAmp DNA mini kit as recommended by the manufacturer (Qiagen)
|
| Label |
Cy3
|
| Label protocol |
10 ng of gDNA were amplified with the processive polymerase phi29 using the GenomiPhi illustrator V2 kit (GE HealthCare, Lifescience, Orsay, France). The amplified gDNA was labeled with the Bioprime CGH Labeling kit (Invitrogen) using d-CTP Cy3/5 fluorochromes (GE HealthCare Lifescience) as recommended by the manufacturer. Labeled amplified gDNA was purified using Pure Link PCR purification columns (Invitrogen).
|
| |
|
| Channel 2 |
| Source name |
NMI
|
| Organism |
Coxiella burnetii |
| Characteristics |
strain: NMI
|
| Growth protocol |
C. burnetii were grown at 35°C on L929 cells using MEM (GIBCO, Invitrogen, Cergy-Pontoise, France) supplemented with 4% SVF (GIBCO) and 1% L-glutamine (GIBCO). Monolayers of cells and the supernatants from three 175 cm² flasks were harvested and incubated with 1% trypsin (GIBCO) for 1 hour at 37°C. Released bacteria were purified from L929 cell debris by differential centrifugation. Purified bacteria were resuspended in 400 µl of PBS and stored at -80°C
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
200 µl of purified bacteria were incubated for 30 min at 70°C with 200 µl of AL lysis buffer (Qiagen, Courtaboeuf, France) and 20 µl of proteinase K (Qiagen). gDNA was extracted and purified using a QiaAmp DNA mini kit as recommended by the manufacturer (Qiagen)
|
| Label |
Cy5
|
| Label protocol |
10 ng of gDNA were amplified with the processive polymerase phi29 using the GenomiPhi illustrator V2 kit (GE HealthCare, Lifescience, Orsay, France). The amplified gDNA was labeled with the Bioprime CGH Labeling kit (Invitrogen) using d-CTP Cy3/5 fluorochromes (GE HealthCare Lifescience) as recommended by the manufacturer. Labeled amplified gDNA was purified using Pure Link PCR purification columns (Invitrogen).
|
| |
|
| |
| Hybridization protocol |
Hybridizations were carried out using two samples of labeled amplified gDNA (150 pmol of each) that were labeled with Cy3 or Cy5 d-CTP. The pooled samples were hybridized using the GE hybridization kit (Agilent Technologies) as recommended by the manufacturer. The mixture was applied to a Surhyb 1 array (Agilent Technologies) and hybridized on the Coxiella burnetii array using an Agilent hybridization chamber (Agilent Technologies). Microarrays were hybridized for 17 h at 62°C in a rotating oven. Microarrays were washed using GE washing buffers (Agilent Technologies), with 5 min of Wash-buffer 1 at room temperature, followed by 1 min of Wash-buffer 2 at 37°C. Microarrays were dried using an acetonitrile bath (VWR, Fontenay sous Bois, France)
|
| Scan protocol |
Microarrays werescanned using a microarray scanner C (Agilent Technologies) with XDR at a 5-µm resolution
|
| Description |
CGH-CB76
Genotyping of Isolate CB76 compare to NMI
|
| Data processing |
The signal intensity and local background were measured for each spot using the array pictures with Feature Extractor software (Agilent Technologies). Data filtering normalizations were obtained using processing signal from obtained data raw extraction using Feature Extractor. Data with low intensity were not included in the analyses.
|
| |
|
| Submission date |
Aug 19, 2011 |
| Last update date |
Dec 31, 2011 |
| Contact name |
LEROY Quentin |
| E-mail(s) |
[email protected]
|
| Organization name |
URMITE
|
| Street address |
27 Boulevard Jean Moulin
|
| City |
Marseille |
| ZIP/Postal code |
13005 |
| Country |
France |
| |
|
| Platform ID |
GPL6675 |
| Series (1) |
| GSE31543 |
Genotyping of Coxiella burnetii strains collection |
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