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| Status |
Public on Jan 26, 2015 |
| Title |
B_cereus UC10070_GSP_2 |
| Sample type |
RNA |
| |
|
| Source name |
RNA from Isolated Bacillus cereus UC10070 spores
|
| Organism |
Bacillus cereus |
| Characteristics |
strain: UC10070
sample type: Germinating Spores
time: T40min
|
| Treatment protocol |
Spores were produced from cells cultured in BP medium (Bacillus Genetic Stock Center, Ohio State University). BP plates were inoculated with 500 μl of B. cereus s.l. UC10070 overnight cultures, and incubated for 4 days at 37°C. Spores were harvested and purified by extensive washing with distilled water at 4°C (Nicholson and Setlow, 1990). The spore crops, checked by phase-contrast microscopy, were free (>99%) of vegetative cells, germinating spores, and debris. Germination assays were carried out at room temperature by inoculating 100 μl of B. cereus. s.l. UC10070 spore suspension (107 CFU/ml) in the selected food products followed by anaerobic packaging obtained using Anaerocult A packs (Merck, Germany) and by a heat treatment for 15 min at 80 °C reproducing retail conditions. The germination process was monitored by optical density measurements (600 nm) at regular time points (every 30 minutes for up to 48 h after heat activation) and by scanning electron microscopy.
|
| Growth protocol |
The strain UC10070 isolated from a biofilm on a spoiled vegetable-based puree was cultured in Brain Heart Infusion (BHI) broth at 37 °C in aerobic conditions on continuous shaking
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from 1 ml of soup for each condition, homogenized with physiological solution, centrifuged at 10000 x g for 10 min and pellets processed using RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. Samples lysis was carried out in a FastPrep instrument (MP Biomedicals) with different settings for spores (SP), germinating spores (GSP) and vegetative cells (C2h and C12h). SP and GSP were processed four times for 50 sec each in the FastPrep machine at setting 6.5 m/s using 0.1 mm zirconia/silica beads (Bio Spec Products, Inc.); C2h and C12h cells were bead-beated one time for 50 sec at 6.5 m/s. Samples were kept on ice during the intermediate processing steps. RNA samples were finally resuspended in 40 μl of RNase-free water and rapidly frozen at -80°C.
|
| Label |
Cy5
|
| Label protocol |
The obtained aRNA, quantified (20 μg per sample) and purified, was labelled with Cy-5 dye; Cy5-aRNA was subsequently purified and its concentration was determined using a spectrophotometer.
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| |
|
| Hybridization protocol |
The fragmented Cy5-aRNA was then mixed with hybridization buffer (6X SSPE, 0.05% Tween-20, 20 mM EDTA, 25% deionised formamide, 0.1 mg/mL sheared salmon sperm DNA and 0.04% SDS) and hybridized to the Combimatrix 12K Chip at 45 °C for 16 h.
|
| Scan protocol |
Scanning was performed by using a ScanArray 4000XL scanner (Perkin-Elmer, USA) following the standard operating protocol.
|
| Description |
Sample name: Germinating Spores T40min 2
Bacillus cereus sl group
This is the second of three biological replicates of germinating spore sample at 40min. These data are derived from the first technical replicate.
|
| Data processing |
Data pre-processing was performed using Combimatrix Blist software (v. 0.6.2.) and Quantile normalization was applied. Subtraction of estimated non-specific binding using the cross-species hybridization data was done.
Hybridization signals from Cy5 channel and median fluorescence intensity
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| |
|
| Submission date |
Jan 23, 2015 |
| Last update date |
Jan 26, 2015 |
| Contact name |
Daniela Bassi |
| E-mail(s) |
daniela.bassi@unicatt.it
|
| Phone |
+390372499108
|
| Organization name |
Università Cattolica del Sacro Cuore
|
| Department |
Istituto di Microbiologia
|
| Lab |
CRB
|
| Street address |
via Milano 24
|
| City |
Cremona |
| State/province |
CR |
| ZIP/Postal code |
26100 |
| Country |
Italy |
| |
|
| Platform ID |
GPL19697 |
| Series (1) |
| GSE65259 |
Study of Bacillus cereus spore germination and outgrowth in a vegetable food model by means of genome-wide transcriptome analysis |
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