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| Status |
Public on Jun 02, 2020 |
| Title |
Ent. faecalis 14 wild type rep.3 |
| Sample type |
RNA |
| |
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| Source name |
Ent. faecalis 14 wild type rep.3
|
| Organism |
Enterococcus faecalis |
| Characteristics |
strain: 14
genotype: wild type
age: 6 hours of growth
|
| Growth protocol |
Cultures of Ent. faecalis 14 and Ent. faecalis 14 ∆Bac mutant and Ent. faecalis 14∆Bac-Comp complemented strains were performed on the M17 medium supplemented with 0.5% (w/v) of glucose (GM17) at 37 °C under semi-aerobic conditions. Data related to genetic approach for mutants engineering as well as their antibacterial potency will be described elsewhere. Growth kinetics were performed on a 96-well microplate by measuring the optical density at a wavelength of 600 nm (OD600) using the SpectraMax i3 spectrophotometer (Molecular Devices, CA, USA). The cultures were inoculated with the same bacterial charge and a measurement points were taken every 30 minutes for 12 h. Bacterial stocks were stored at - 80°C in GM17 broth supplemented with 50% (v/v) glycerol.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For microarray analysis, three distinct cultures of Ent. faecalis 14 ∆Bac were used (ΔBac1, ΔBac2 and ΔBac3) in order to carry comparisons with Ent. faecalis 14 (WT1, WT2 and WT3), after 6 h of growth in GM17 medium respectively. Analyses were performed with total RNA isolated using NucleoSpinTM RNA Plus columns (Macherey-Nagel, Hoerdt, France). RNA quality was observed with Nanodrop and absorbance ratios A260/280 and A260/230 were found between 2.0 and 2.2. RNA quality was also examined with Bioanalyzer 2100 (Agilent, Les Ulis, France) and a minimal RNA integrity number (RIN) of 0.8 was required for all samples.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
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| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Mus musculus Sure Print GE 4x44 v2 microarrays with oligonucleotide 45,220 probes (G2519F) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
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| Scan protocol |
Slides 8x15K were scanned immediately after washing on the Scanner GenePix 4200B (Molecular Device) using one color scan setting
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| Description |
Gene expression after 6h of culture
|
| Data processing |
The scanned images were analyzed with GenePix Pro Software 6.0 (Molecular Device) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
Slides were 8-plex and only 1 or 2 blocks by slide concerned this project. In the title of the gpr file, the block of interest is indicated. Raw data .xls files include raw data in the block used in this study.
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| |
|
| Submission date |
May 05, 2020 |
| Last update date |
Jun 03, 2020 |
| Contact name |
Anca Lucau-Danila |
| Organization name |
University of Lille
|
| Department |
Biology
|
| Lab |
Charles Viollette Institute
|
| Street address |
Cité Scientifique SN2 303
|
| City |
Villeneuve d'Ascq |
| ZIP/Postal code |
59655 |
| Country |
France |
| |
|
| Platform ID |
GPL28480 |
| Series (1) |
| GSE149873 |
Gene expression profiles of differentially expressed genes in Enterococcus faecalis 14 ΔBac mutant strain |
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