|
| Status |
Public on Apr 20, 2023 |
| Title |
Ent. faecalis 14 delta pnpA rep.3 [P3-3h] |
| Sample type |
RNA |
| |
|
| Source name |
Ent. faecalis 14 delta pnpA-3h
|
| Organism |
Enterococcus faecalis |
| Characteristics |
strain background: 14
genotype: delta pnpA
age: 3 hours
|
| Treatment protocol |
The ∆pnpA mutant strain was constructed by allelic exchange using a method based on the conditional replication of the pLT06 vector.
|
| Growth protocol |
Cultures of E. faecalis strains were performed in M17 medium supplemented with glucose at 0.5% (w/v) to obtain the GM17, or in brain heart infusion (BHI), under semi-aerobic condition at 37°C. When necessary, media were supplemented with erythromycin (Em) (150 µg/mL) or chloramphenicol (Cm) (15 µg/mL). Growth of E. faecalis cultures was followed by measuring the optical density at a wavelength of 600 nm (OD600) with a spectrophotometer (Aquoalabo, France), and by determining cfu counts on agar plates. E. coli strains were cultivated at 37°C in Luria-Bertani (LB) broth with shaking (160 rpm) or on LB agar medium. When appropriate, erythromycin (150 µg/mL) or chloramphenicol (10 µg/mL) or ampicillin (100µ/mL) was added to the medium. Bacterial stocks were stored at - 80°C in GM17, BHI or LB broth supplemented with 50% (v/v) glycerol.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For microarray analysis, three distinct cultures of Ent. faecalis 14 ∆pnpA were used (P1, P2 and P3) in order to carry comparisons with Ent. faecalis 14 (WT1, WT2 and WT3), after 3h and after 6h 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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| |
|
| 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-086007 EfaecalisDD14_V2 microarrays 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.
|
| Scan protocol |
Slides 8x15K were scanned immediately after washing on the Scanner GenePix 4200B (Molecular Device) using one color scan setting
|
| Description |
Gene expression after 3h 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.
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| |
|
| Submission date |
Jul 19, 2021 |
| Last update date |
Apr 20, 2023 |
| 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) |
| GSE180397 |
Gene expression profiles of differentially expressed genes in Enterococcus faecalis 14 ΔpnpA mutant strain |
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