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Status |
Public on Mar 29, 2017 |
Title |
SL1344_Exponential_BR1 |
Sample type |
mixed |
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|
Channel 1 |
Source name |
SL1344 cells
|
Organism |
Salmonella enterica subsp. enterica serovar Typhimurium |
Characteristics |
strain: SL1344
|
Growth protocol |
Cultures of S. Typhimurium SL1344 were inoculated 1:100 into 25 mls fresh Lennox broth and grown at 37oC until OD600 0.2 (Late exponential phase) or OD600 2.0 (Early stationary phase) was reached
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was extracted from the cells using the SV total RNA isolation kit from promega as described (www.ifr.ac.uk/safety/molmicro). Sonicated genomic DNA was used as a reference in these experiments and was purified by standard phenol-chloroform extraction followed by sodium acetate/ethanol precipitation.
|
Label |
Cy3
|
Label protocol |
cDNA synthesis was performed with the Superscript double-stranded cDNA synthesis kit (Invitrogen), following the random-priming method outlined by the manufacturer. Fluorescent labeling of DNA samples for transcriptomic experiments were carried out using the BioPrime Random Labeling kit (Invitrogen) as follows: 60 ml 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/ ml). *[1000 ng of cDNA and 1000 ng of sonicated genomic DNA were fluorescently labeled with Cy3 d-CTP and Cy5-dCTP respectively]. The Labeling reactions wereas carried out at 37 oC overnight and 15 ul of stop buffer added to terminate the reaction.
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|
|
Channel 2 |
Source name |
SL1344 control DNA
|
Organism |
Salmonella enterica subsp. enterica serovar Typhimurium |
Characteristics |
strain: SL1344
|
Growth protocol |
Cultures of S. Typhimurium SL1344 were inoculated 1:100 into 25 mls fresh Lennox broth and grown at 37oC until OD600 0.2 (Late exponential phase) or OD600 2.0 (Early stationary phase) was reached
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Total RNA was extracted from the cells using the SV total RNA isolation kit from promega as described (www.ifr.ac.uk/safety/molmicro). Sonicated genomic DNA was used as a reference in these experiments and was purified by standard phenol-chloroform extraction followed by sodium acetate/ethanol precipitation.
|
Label |
Cy5
|
Label protocol |
cDNA synthesis was performed with the Superscript double-stranded cDNA synthesis kit (Invitrogen), following the random-priming method outlined by the manufacturer. Fluorescent labeling of DNA samples for transcriptomic experiments were carried out using the BioPrime Random Labeling kit (Invitrogen) as follows: 60 ml 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/ ml). *[1000 ng of cDNA and 1000 ng of sonicated genomic DNA were fluorescently labeled with Cy3 d-CTP and Cy5-dCTP respectively]. The Labeling reactions wereas carried out at 37 oC overnight and 15 ul of stop buffer added to terminate the reaction.
|
|
|
|
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 an Agilent G2505C scanner. Cy3 and Cy5 images were acquired at 5 micron resolution. Scanned images were analyzed using Feature extraction software (Agilent).
|
Description |
biological replicate 1
|
Data processing |
median normalization of background subtracted Cy3/Cy5 ratios
|
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Submission date |
Mar 28, 2017 |
Last update date |
Mar 29, 2017 |
Contact name |
Shane Dillon |
E-mail(s) |
[email protected]
|
Organization name |
Dublin Institute of Technology
|
Department |
School of Biological Sciences
|
Street address |
Kevin Street
|
City |
Dublin |
ZIP/Postal code |
Dublin 8 |
Country |
Ireland |
|
|
Platform ID |
GPL11416 |
Series (1) |
GSE97161 |
Salmonella enterica serovar Typhimurium gene expression in exponential and stationary phase cultures |
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