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| Status |
Public on Dec 01, 2017 |
| Title |
gDNA_Fsuccinogenes_S85 |
| Sample type |
genomic |
| |
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| Source name |
Bacteria
|
| Organism |
Fibrobacter succinogenes subsp. succinogenes S85 |
| Characteristics |
gut origin: Rumen
|
| Treatment protocol |
No treatment
|
| Growth protocol |
Genomic DNA was extracted from 4 bacteria strains. Strains Fibrobacter succinogenes S85 (ATCC 19169), Ruminococcus albus 20 (ATCC 27211) and Bacteroides xylanisolvens XB1A (DSM 18836T) were grown 15 h under strictly anaerobic conditions (Hungate, 1950) in a complex medium containing 20% of clarified rumen fluid (Béra-Maillet et al., 2000) and 0.2 g of cellobiose (Sigma-Aldrich) as carbon source, at 39°C for the two first ones and 37°C for the third one. The strain Escherichia coli K12 (ATCC 10798) was cultivated 15 h at 37°C in Luria-Bertani medium under shaking and aerobic conditions. (
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Culture of bacterial strains (10 mL) were centrifuged 20 min at 10,000 g and bacterial pellets were washed 3 times with 2 mL of phosphate buffered saline (PBS). DNA extraction was performed using the EasyDNA kit (Invitrogen) following the manufacturer instructions slightly modified. Briefly, the bacterial pellet was incubated 5 min in 200 μL of PBS and 1 μL of protéinase K (20 mg/mL, Invitrogen). Then, bacteria were grinded 3 min with ~100 mg of 0.1 mm zirconia beads (BioSpec Products) using the model MM2000 BeadBeater instrument (Retsch). Concentration and quality of isolated gDNA were checked with the spectrophotometer Nanodrop 1000 (Thermo Fisher Scientific). Their integrity was verified by 1% agarose gel electrophoresis. gDNA were kept at -20°C.
|
| Label |
Cy5
|
| Label protocol |
Genomic DNAs from each bacterial strain (2 µg) were sheared in a 200 μL milliQ water volume by 10 sec of sonication (Vibra Cell VC300, Sonics & Materials). The length of sheared gDNA (range between 500 bp and 1500 bp) was checked by 1% agarose gel electrophoresis. Fragmented gDNA was labelled using the Bioprime Total Genomic Labelling kit (Invitrogen) and Alexa Fluor 3 or 5 dyes following the supplier’s instructions. A degree of labelling higher than 0.7 for the Alexa fluor 3 and 1.2 for the Alexa fluor 5 was considered as acceptable.
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| |
|
| Hybridization protocol |
DNA hybridization (1 μg for each DNA) was performed at 65°C during 24 h following the Agilent protocol ‘Oligonucleotide Array-Based CGH for Genomic DNA Analysis’.
|
| Scan protocol |
Immediately after washing steps, microarray slides were scanned using an Agilent G2505B microarray scanner (Agilent Technologies) with a resolution of 5 μm and the XDR mode (Extended Dynamic Range) set up at 10% and 100% PMT gain.
|
| Description |
gDNA
|
| Data processing |
Fluorescence intensities of oligonucleotide spots were extracted from the scanned images using the Feature Extraction software (V11.0, Agilent Technologies). Then data were processed with dedicated scripts based on C++ and Delphi languages. For each probe, median intensity value of the 3 replicates was conserved and used as the probe signal value. The SNR (Signal-to-Noise Ratio), similar to the detection threshold response (positive hybridization) and corresponding to the probe signal value divided by the local background intensity value, was calculated for each probe. The SNR thresholds were set to 3 for 25-mer probes and 6 for 54-mer probes. Such SNR threshold values ensured a very specific response of the FibroChip, except for genes belonging to very close strains from the same species. Results for 25- and 54-mer probes were treated independently and for each probe type, a gene was considered as detected when 65% of probes were positive. The SNR value of a detected gene was then calculated by the mean of the SNR of all 25- or 54-mer probes targeting this gene, meeting or not the defined SNR threshold.
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| |
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| Submission date |
Nov 30, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Sophie Comtet-Marre |
| Organization name |
Université Clermont Auvergne, INRA
|
| Lab |
UMR 454 MEDIS
|
| Street address |
Centre Auvergne-Rhône-Alpes
|
| City |
Saint-Genès-Champanelle |
| ZIP/Postal code |
63122 |
| Country |
France |
| |
|
| Platform ID |
GPL24327 |
| Series (1) |
| GSE107550 |
The FibroChip, a functional DNA microarray to monitor cellulolysis and hemicellulolysis activities of rumen microbiota |
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