|
| Status |
Public on Nov 09, 2013 |
| Title |
SL1344_wildtype_BR1 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
RNA_SL1344 cells grown to OD600nm 0.3
|
| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium |
| Characteristics |
strain: SL1344 wild type
|
| Growth protocol |
SL1344 cells were inoculated 1:100 into 25 ml fresh Luria Broth (LB) and grown at 37degrees celsius in a shaking waterbath at 200 rpm until OD600 0.3
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For transcriptome experiments 2.0 OD600nm units of bacterial cultures were harvested during mid-exponential growth (OD600nm= 0.3). Total RNA was extracted as above using a Promega SV Total RNA Isolation kit according to manufacturer guidelines. RNA samples were quantified using a Nanodrop ND-1000 and checked for purity and degradation by gel electrophoresis. Total RNA samples were then converted to double stranded cDNA pools in a two-step process using a Superscript™ Double-Stranded cDNA synthesis kit (Invitrogen) according to manufacturer guidelines
|
| Label |
Cy3 dCTP
|
| Label protocol |
Fluorescent labeling of genomic DNA and cDNA samples were carried out using the BioPrime Random Labeling kit (Invitrogen) as follows: 60 ul of 2.5 x random primer solution, x* ul DNA, and (70.5-x) ul sterile water were heated at 100oC for 10 minutes, snap chilled on ice and following were then added – 15 ul dNTP mix (1mM dCTP, 2mM dGTP, dTTP, dATP), 1.5 ul 1 mM Cy3/Cy5 labeled dCTP (GE healthcare) and 3 ul Klenow fragment (40 U/ ul). *[200 ng of cDNA and 200 ng of Input DNA were fluorescently labeled with Cy3 d-CTP and Cy5-dCTP respectively]. The Labeling reactions wereas carried out at 37 oC overnight and 15 ml of stop buffer added to terminate the reaction. Labeled DNAs were purified using G-50 columns (GE-healthcare), according to manufacturers instructions.
|
| |
|
| Channel 2 |
| Source name |
genomic DNA_SL1344 cells grown to OD600nm 0.3
|
| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium |
| Characteristics |
strain: SL1344
|
| Growth protocol |
SL1344 cells were inoculated 1:100 into 25 ml fresh Luria Broth (LB) and grown at 37degrees celsius in a shaking waterbath at 200 rpm until OD600 0.3
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For transcriptome experiments 2.0 OD600nm units of bacterial cultures were harvested during mid-exponential growth (OD600nm= 0.3). Total RNA was extracted as above using a Promega SV Total RNA Isolation kit according to manufacturer guidelines. RNA samples were quantified using a Nanodrop ND-1000 and checked for purity and degradation by gel electrophoresis. Total RNA samples were then converted to double stranded cDNA pools in a two-step process using a Superscript™ Double-Stranded cDNA synthesis kit (Invitrogen) according to manufacturer guidelines
|
| Label |
Cy5 dCTP
|
| Label protocol |
Fluorescent labeling of genomic DNA and cDNA samples were carried out using the BioPrime Random Labeling kit (Invitrogen) as follows: 60 ul of 2.5 x random primer solution, x* ul DNA, and (70.5-x) ul sterile water were heated at 100oC for 10 minutes, snap chilled on ice and following were then added – 15 ul dNTP mix (1mM dCTP, 2mM dGTP, dTTP, dATP), 1.5 ul 1 mM Cy3/Cy5 labeled dCTP (GE healthcare) and 3 ul Klenow fragment (40 U/ ul). *[200 ng of cDNA and 200 ng of Input DNA were fluorescently labeled with Cy3 d-CTP and Cy5-dCTP respectively]. The Labeling reactions wereas carried out at 37 oC overnight and 15 ml of stop buffer added to terminate the reaction. Labeled DNAs were purified using G-50 columns (GE-healthcare), according to manufacturers instructions.
|
| |
|
| |
| Hybridization protocol |
Cy3 labeled cDNA and Cy5 labeled control genomic DNAs were co-precipitated using standard sodium acetate/ethanol procedures and resuspended in hybridization buffer (Oxford Gene Technologies), and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers and hybridiized for 24 hours at 55oC in an Agilent hybiridization oven at 5 rpm. After hybridization, slides were washed according to instructions provided by Oxford Gene Technologies
|
| Scan protocol |
The microarray slides were scanned using a GenePix 4000B scanner (Axon Instruments). Cy3 and Cy5 images were acquired at 5 micron resolution.
|
| Description |
Sample 1; biological replicate 1
|
| Data processing |
Quantification of fluorescent spot intensities and local background data was performed using the GenePix 3.0 software supplied. The Cy3/Cy5 ratio and median Cy3/Cy5 ratio were determined for each probe. Cy3/Cy5 values were median normalised and the intensities of the multiple probes across each gene were averaged
|
| |
|
| Submission date |
Nov 08, 2013 |
| Last update date |
Nov 09, 2013 |
| Contact name |
Shane Dillon |
| E-mail(s) |
[email protected]
|
| Organization name |
Moyne Institute of Preventive Medicine
|
| Department |
Department of Microbiology
|
| Street address |
Trinity College
|
| City |
Dublin |
| ZIP/Postal code |
Dublin 2 |
| Country |
Ireland |
| |
|
| Platform ID |
GPL10008 |
| Series (1) |
| GSE52235 |
Gene expression analysis of Salmonella enterica serovar Typhimurium strain SL1344 swap |
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