|
| Status |
Public on Dec 01, 2011 |
| Title |
CIRM1_4°C_3days_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
strain CIRM1, 4°C, 3 days, repetition 2
|
| Organism |
Propionibacterium freudenreichii subsp. shermanii |
| Characteristics |
strain: CIRM-BIA1 (CIP103027)
|
| Growth protocol |
Bacteria were grown in YEL broth under air atmosphere without agitation. Cultures were incubated at 30°C until they reached an OD650 of about 2. Then, cultures were incubated for a further 9 days at 4°C. Lactate was added after 24 h of incubation at 4°C in order to prevent carbon starvation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested (8000 × g, 10 min, ambient temperature or 4°C regarding the sampling time). Cell pellets were suspended in 100 µl of TE buffer 1x (Qiagen, Courtaboeuf, France) containing lysozyme (20 mg/ml), and incubated for 15 min at 24 °C before RLT buffer (from the Qiagen RNeasy minikit) addition at a ratio of 35/10 (vol/vol). 50 mg of zirconium beads and 100 µl of sodium dodecyl sulfate (SDS, 20%) were then added and cells were disrupted for 2 × 90 seconds at 30 Hz by using a Retsch MM301 high-speed mixer mill (Grosseron, France). RNA was extracted according to the instructions of the RNeasy kit. Then DNase treatments of the RNA samples were performed using a DNA-free kit (Ambion, Texas, USA) according to the manufacturer’s instructions. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from (50-100 ng) RNA using the Low Input Quick Amp Labeling 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.
|
| |
|
| Hybridization protocol |
600 ng 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 25µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer instructions. On completion of the fragmentation reaction, 25µl of 2x Hi-RPM hybridization buffer (Agilent) was added to the fragmentation mixture and hybridized to Agilent specific 8x15K microarray 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 for 1 minute with 37°C GE Wash buffer 2 (Agilent), and terminal 1 min at room temperature with Acetonetril.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565CA) using one color scan setting for 8x15K array slides (Scan Area 61x21.6 mm, Scan resolution 5µM, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
3DB
Gene expression after 3 days at 4°C
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters ( GE1_105_Dec08 and Grid: array specific) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Jul 21, 2011 |
| Last update date |
Dec 01, 2011 |
| Contact name |
Marion Dalmasso |
| E-mail(s) |
marion.dalmasso@teagasc.ie
|
| Organization name |
Teagasc
|
| Department |
Food Safety
|
| Lab |
Kieran Jordan Lab
|
| Street address |
Moorepark
|
| City |
Fermoy |
| ZIP/Postal code |
Co. Cork |
| Country |
Ireland |
| |
|
| Platform ID |
GPL13959 |
| Series (1) |
| GSE30841 |
Propionibacterium freudenreichii transcriptomic response during cold storage |
|
