|
| Status |
Public on Jan 31, 2012 |
| Title |
WTvsKOcomX CDML replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
LMD-9WT CDML OD 0.4
|
| Organism |
Streptococcus thermophilus |
| Characteristics |
strain: LMD-9
isolate: clone 1
|
| Growth protocol |
Strains LMD-9 WT and CB003 were grown at 37°C during 16h in M17 medium supplemented with lactose 1% (M17L), washed twice (5,000 x g, 9 min, room temperature) in one volume of CDM medium supplemented with lactose 1% (CDML) and resuspended in one volume of CDML. Cultures were then 30-fold diluted in CDML. When cells reached the mid-log phase (OD600 of 0.4), 25-ml aliquots were collected for total RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested by centrifugation (7,000 x g for 4 min) and mechanically broken with 0.18-mm-diameter glass beads in a Braun Homogenizer (three 1-min periods of homogenization with 1-min intervals on ice). Total RNA was extracted using the High Pure RNA isolation kit (ROCHE, Basel, Switzerland)
|
| Label |
Alexa Fluor 555
|
| Label protocol |
First strand cDNA was synthetized from 10 microg RNA. Synthesized cDNA were purified and labeled with Alexa Fluor 555 and Alexa Fluor 647. Experiments were carried out according to instructions of the Superscript Plus Indirect cDNA Labeling system (Invitrogen).
The quality of the labeled cDNA and its concentration were respectively measured with the 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA) and ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE).
|
| |
|
| Channel 2 |
| Source name |
KOcomX CDML OD 0.4
|
| Organism |
Streptococcus thermophilus |
| Characteristics |
isolate: clone 1
strain: CB003
|
| Growth protocol |
Strains LMD-9 WT and CB003 were grown at 37°C during 16h in M17 medium supplemented with lactose 1% (M17L), washed twice (5,000 x g, 9 min, room temperature) in one volume of CDM medium supplemented with lactose 1% (CDML) and resuspended in one volume of CDML. Cultures were then 30-fold diluted in CDML. When cells reached the mid-log phase (OD600 of 0.4), 25-ml aliquots were collected for total RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested by centrifugation (7,000 x g for 4 min) and mechanically broken with 0.18-mm-diameter glass beads in a Braun Homogenizer (three 1-min periods of homogenization with 1-min intervals on ice). Total RNA was extracted using the High Pure RNA isolation kit (ROCHE, Basel, Switzerland)
|
| Label |
Alexa Fluor 647
|
| Label protocol |
First strand cDNA was synthetized from 10 microg RNA. Synthesized cDNA were purified and labeled with Alexa Fluor 555 and Alexa Fluor 647. Experiments were carried out according to instructions of the Superscript Plus Indirect cDNA Labeling system (Invitrogen).
The quality of the labeled cDNA and its concentration were respectively measured with the 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA) and ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE).
|
| |
|
| |
| Hybridization protocol |
Hybridization of the labeled cDNA (0.3 microg per sample) were added to hybridization buffer (Agilent In Situ Hybridization Kit Plus) and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequentially according to Agilent instructions.
|
| Scan protocol |
Scanned on an Agilent Scanner G2565B
Images were quantified using Agilent Feature Extraction Software (version 9.1).
|
| Description |
Biological replicate 1 of 2, dye swap
|
| Data processing |
Agilent Feature Extraction Software (v 9.1) was used for background subtraction and LOWESS normalization.
|
| |
|
| Submission date |
Apr 04, 2011 |
| Last update date |
Feb 01, 2012 |
| Contact name |
Pascal Hols |
| E-mail(s) |
pascal.hols@uclouvain.be
|
| Phone |
+32 10 47 88 96
|
| Fax |
+32 10 47 31 09
|
| Organization name |
Université catholique de louvain
|
| Department |
Institut des Sciences de la Vie
|
| Lab |
Biochimie et Génétique Moléculaire Bactérienne
|
| Street address |
Place Croix du Sud 5
|
| City |
Louvain-La-Neuve |
| ZIP/Postal code |
1348 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL13365 |
| Series (1) |
|
