|
| Status |
Public on Oct 28, 2009 |
| Title |
WT_antiFLAG_BR2 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
WT_antiFLAG_BR2
|
| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344 |
| Characteristics |
antibody: anti-FLAG
genotype/variation: wild type
strain: Salmonella enterica serovar Typhimurium SL1344
|
| Growth protocol |
Over-night cultures were diluted 1000-fold into 250ml flasks containing 25ml of LB and grown at 37oC under aeration for 160min to reach the mid-exponential phase of growth.
|
| Extracted molecule |
other |
| Extraction protocol |
After reaching early exponential growth (OD600 0.12), formaldehyde was added to reach a 1% final concentration and the cultures incubated for 15 min at 37oC. The cross-linking was then stopped by adding 1/4 vol. 1M glycine. Cells were washed three times in ice-cold PBS (pH7.4), resuspended in 500ul lysis buffer (10mM Tris pH8.0; 50mM NaCl; 10mM EDTA; 20% sucrose; 20 mg/ml lysozyme) and incubated 45 min at 37oC at which point 500ul 2x RIPA (100mM Tris pH8.0; 300mM NaCl; 2% Nonidet P40; 1% sodium deoxycholate; 0.2% SDS) was added. Chromatin was solubilised by sonication until DNA fragments were between 300 and 750 bp, and the lysate centrifuged at 12000g for 10 min to remove debris. Cell extracts were then incubated with the appropriate antibody for 4h at 4oC. After the incubation of the cell extracts with the antibody, 50ul protein G beads were added and left for 16h at 4oC. The protein G beads were then washed twice in 1xRIPA, twice in wash solution (10mM Tris pH8.0; 250mM LiCl; 1mM EDTA; 0.5% Nonidet P40; 0.5% sodium deoxycholate) and twice in TE (10mM Tris pH8.0; 1mM EDTA). The immunoprecipitate was eluted in 150ul of elution buffer (50mM Tris pH8.0; 10mM EDTA; 1% SDS) prewarmed at 65oC. Cross-linking was reversed by incubating the eluate in 0.5x elution buffer containing 0.8mg/ml pronase (Sigma) and DNA purified using the Qiagen PCR purification kit.
|
| Label |
Cy3
|
| Label protocol |
1/5 of the immunoprecipitated DNA was labelled using the 'Direct labelling of DNA' protocol that can be found at http://www.ifr.bbsrc.ac.uk/safety/microarrays/protocols.html.
|
| |
|
| Channel 2 |
| Source name |
Reference genomic DNA
|
| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344 |
| Characteristics |
strain: 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 |
Cy5
|
| Label protocol |
Labelling protocol at http://www.ifr.ac.uk/safety/Microarrays/protocols.html
|
| |
|
| |
| 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 10um resolution scan & see http://www.ifr.ac.uk/safety/Microarrays/default.html#protocols
|
| Description |
Total array is 25 blocks arranged in 5X5 configuration.
|
| Data processing |
Intensity ratios of channel 1 (coIP-DNA) divided by channel 2 (reference gDNA) signals are calculated using BlueFuse 3.1 (BlueGnome, Cambridge, UK). Data centring was subsequently performed using the block median.
|
| |
|
| Submission date |
Oct 07, 2009 |
| Last update date |
Oct 28, 2009 |
| 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) |
| GSE18450 |
Identification of StpA-binding sites on the Salmonella genome |
|
