|
| Status |
Public on Apr 17, 2007 |
| Title |
spxB 164880 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
L.lactis MG1363 VES3910
|
| Organism |
Lactococcus cremoris subsp. cremoris MG1363 |
| Characteristics |
VES3910
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from 50 ml of cells grown to midexponential phase of growth (A600 = 0.3) in M17 containing 0.25% glucose (GM17). Cells were harvested by centrifugation for 1 min at 10,000 rpm at room temperature. Cell pellets were immediately frozen in liquid nitrogen and stored at –80 °C. Pellets were resuspended in 500 µl of 10 mM Tris-HCl, 1 mM EDTA (T10E1), pH 8.0, after which 50 µl of 10% SDS, 500 µl of phenol/chloroform, 500 mg of glass beads (75–150 µm), and 175 µl of Macaloid suspension (Bentone) were added. Furter purification was done using the High Pure RNA isolation, using the suppliers protocol.
|
| Label |
cy3
|
| Label protocol |
Synthesis of cDNA and indirect Cy-3/Cy-5-dCTPs labeling of 15–20 µg of total RNA was performed with the CyScribe Post labeling kit (Amersham Biosciences) according to the supplier's instructions.
|
| |
|
| Channel 2 |
| Source name |
L.lactis MG1363 VES4075
|
| Organism |
Lactococcus cremoris subsp. cremoris MG1363 |
| Characteristics |
VES4075
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from 50 ml of cells grown to midexponential phase of growth (A600 = 0.3) in M17 containing 0.25% glucose (GM17). Cells were harvested by centrifugation for 1 min at 10,000 rpm at room temperature. Cell pellets were immediately frozen in liquid nitrogen and stored at –80 °C. Pellets were resuspended in 500 µl of 10 mM Tris-HCl, 1 mM EDTA (T10E1), pH 8.0, after which 50 µl of 10% SDS, 500 µl of phenol/chloroform, 500 mg of glass beads (75–150 µm), and 175 µl of Macaloid suspension (Bentone) were added. Furter purification was done using the High Pure RNA isolation, using the suppliers protocol.
|
| Label |
cy5
|
| Label protocol |
Synthesis of cDNA and indirect Cy-3/Cy-5-dCTPs labeling of 15–20 µg of total RNA was performed with the CyScribe Post labeling kit (Amersham Biosciences) according to the supplier's instructions.
|
| |
|
| |
| Hybridization protocol |
Hybridization (16 h at 45 °C) of labeled cDNA was performed in Ambion Slidehyb #1 hybridization buffer (Ambion Europe) on superamine glass slides (Array-It; SMMBC), containing technical replicates.
|
| Scan protocol |
Slides were scanned using the Genepix 4200AL DNA micro array slide scanner.
Quantification of the signal of the spots on slides was done with ArrayPro 4.5.
|
| Description |
see article SpxB regulates O-acetylation dependent resistance of Lactococcus lactis peptidoglycan to hydrolysis; submitted for publication
|
| Data processing |
Slide data were processed and normalized using MicroPreP, and CyberT implementation of the Student's t test (ref. van Hijum, S. A., J. Garcia de la Nava, O. Trelles, J. Kok, and O. P. Kuipers. 2003. MicroPreP: a cDNA microarray data pre-processing framework. Appl. Bioinformatics 2:241-244.).
|
| |
|
| Submission date |
Mar 28, 2007 |
| Last update date |
Apr 16, 2007 |
| Contact name |
Anne de Jong |
| E-mail(s) |
[email protected]
|
| Phone |
+31 50 363 2047
|
| Organization name |
university of Groningen
|
| Department |
Molecular Genetics
|
| Street address |
Nijenborgh 7
|
| City |
Groningen |
| ZIP/Postal code |
9747 AG |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL5048 |
| Series (1) |
| GSE7386 |
SpxB regulates O-acetylation dependent resistance of Lactococcus lactis peptidoglycan to hydrolysis |
|
