|
| Status |
Public on Aug 23, 2008 |
| Title |
HFQ::FLAG_coIP_ESP_2 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
Reference genomic DNA
|
| Organism |
Salmonella enterica |
| Characteristics |
Salmonella enterica serovar Typhimurium SL1344
|
| Growth protocol |
S. Typhimurium grown to stationary phase in LB-broth
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA isolated using the Qiagen 'Genomic DNA' Kit (Cat. No.: 19060 for the buffer kit; 10243 for the columns)
|
| Label |
Cy3
|
| Label protocol |
Labelling protocol at http://www.ifr.ac.uk/safety/Microarrays/protocols.html
|
| |
|
| Channel 2 |
| Source name |
Hfq3xFLAG-RNA coimmunoprecipitate
|
| Organism |
Salmonella enterica |
| Characteristics |
Salmonella enterica serovar Typhimurium SL1344 hfq3xFLAG
|
| Extracted molecule |
other |
| Extraction protocol |
Strains were grown in L-broth under normal aeration at 37°C up to ESP (OD600 = 2.0). Coimmunoprecipitated RNA was isolated following the protocol described in: Pfeiffer et al. (2007); Molecular microbiology 66, 1174-1191.
|
| Label |
Cy5
|
| Label protocol |
100-200 ng immunoprecipitated RNA was reverse transcribed into ssDNA by random priming using 200 U of AffinityScript (Stratagene) at 50°C. RNA was subsequently hydrolysed by adding 0.1N NaOH and incubating the sample for 10min at 70°. The alkali was neutralised adding 0.1N HCl. As all the nucleotide probes present on the microarrays used (Platform GPL5791) correspond to the ‘+’-strand, prior to the incorporation of the fluorescent label we converted ssDNA to dsDNA using random priming and the klenow fragment from the BioPrime DNA labelling system (Invitrogen). Cy-dye incorporation into cDNA was performed using the 'Direct labelling of DNA' protocol that can be found at http://www.ifr.bbsrc.ac.uk/safety/microarrays/protocols.html. All clean up steps were performed by ethanol precipitation rather than by using spin columns to avoid loosing the shorter RNA/cDNA species.
|
| |
|
| |
| Hybridization protocol |
Labelled samples were resuspended in hybridization buffer (1 M NaCl; 50 mM Mes, pH 7.0; 20% formamide; 1% Triton X-100). The hybridization was performed in microarray chambers (Agilent Technologies) and rotated at 55° over 48 h. The arrays were then washed with Wash 1 [6× standard saline phosphate/EDTA (0.18 M NaCl/10 mM phosphate, pH 7.4/1 mM EDTA] (SSPE)/0.005% N-lauryl sarcosine] and Wash 2 (0.06% SSPE/0.18% polyethylene glycol 200), both for 5 min at room temperature.
|
| Scan protocol |
Axon GenePix Autoloader 4200AL 10nm resolution scan & see http://www.ifr.ac.uk/safety/Microarrays/default.html#protocols
|
| Description |
Intensity ratios of labelled coIP-RNA (channel 2) divided by DNA (channel 1) signals minus respective background signals. Total array is 25 blocks arranged in 5X5 configuration.
|
| Data processing |
Image analysis was performed using the BlueFuse software (BlueGnome). Block-median data centring was then performed in BlueFuse to obtain a median ratio (Cy5/Cy3) for each of the 25 blocks equal to 1. This step generated the AMPCH1 and AMPCH2 values. When comparing AMPCH1 and AMPCH2 values from different hybridisations, we observed slight deviations which were dependent on gene expression levels. These were corrected using the Loess function in R (Limma package), thus generating values found in the VALUE column.
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| |
|
| Submission date |
Jan 11, 2008 |
| Last update date |
Aug 23, 2008 |
| Contact name |
Sacha Lucchini |
| E-mail(s) |
sacha.lucchini@bbsrc.ac.uk
|
| Organization name |
Institute of Food Research
|
| Department |
Molecular Microbiology
|
| Street address |
Colney Lane
|
| City |
Norwich |
| ZIP/Postal code |
NR4 7UA |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL5791 |
| Series (1) |
| GSE10149 |
Identification of RNA molecules associated with the RNA-chaperone Hfq |
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