|
| Status |
Public on Jan 06, 2015 |
| Title |
Zur 2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Control DNA replicate 2
|
| Organism |
Bacillus subtilis subsp. subtilis str. 168 |
| Characteristics |
genotype: zur::zur-spa strain
sample type: whole-cell extract DNA
antibody: none
|
| Growth protocol |
Cells were grown in a flask of 2 L LB medium until OD600nm reached 0.6.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
0.6 % formaldehyde was added in the flasks for 20 min at room temperature before quenching with 125 mM glycine. Cells were harvested by centrifugation at 6000g for 10 min, washed twice with 200 mL of buffer A (10 mM Tris-HCl pH=7.5, 150 mM NaCl), and then lysed in 12 mL of buffer B (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2 mM EDTA, 0.1 % (v/v) TritonX-100) supplemented with 0.1 mg/mL RNase A and 1 mg/mL lysozyme for 15 min at 37°C. DNA was then sheared by sonication in order to obtain 0.2 to 1 kb fragments. An aliquot of 50 µL of the whole cell extract (WCE) volume was saved for use as control DNA. The rest was used for immuno-precipitation DNA (IP): 400 μL of anti-FLAG M2 agarose beads (Sigma A2220) were incubated 2 h with cell lysates under gentle agitation. Then IP beads were washed 3 times in buffer B before being resuspended into 500 µL of buffer C (100 mM Tris-HCl pH=8, 100 g/L, 1% SDS, 10 mM EDTA) to reverse cross-linking for 18 h at 65°C. WCE and IP DNA fragments were recovered and purified using QIAquick PCR purification kit (Qiagen). Remaining RNA was removed by RNase A treatment followed by a second DNA purification step.
|
| Label |
Cy3
|
| Label protocol |
Labelling was done by Roche Nimblegen (Madison, WI, USA) using the standard Nimblegen protocol. ChIP DNA was labelled with Cy5 and control DNA was labelled with Cy3.
|
| |
|
| Channel 2 |
| Source name |
Immunoprecipitated DNA replicate B
|
| Organism |
Bacillus subtilis subsp. subtilis str. 168 |
| Characteristics |
genotype: zur::zur-spa strain
sample type: Immunoprecipitated DNA
antibody: anti-FLAG M2 (Sigma A2220)
|
| Growth protocol |
Cells were grown in a flask of 2 L LB medium until OD600nm reached 0.6.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
0.6 % formaldehyde was added in the flasks for 20 min at room temperature before quenching with 125 mM glycine. Cells were harvested by centrifugation at 6000g for 10 min, washed twice with 200 mL of buffer A (10 mM Tris-HCl pH=7.5, 150 mM NaCl), and then lysed in 12 mL of buffer B (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2 mM EDTA, 0.1 % (v/v) TritonX-100) supplemented with 0.1 mg/mL RNase A and 1 mg/mL lysozyme for 15 min at 37°C. DNA was then sheared by sonication in order to obtain 0.2 to 1 kb fragments. An aliquot of 50 µL of the whole cell extract (WCE) volume was saved for use as control DNA. The rest was used for immuno-precipitation DNA (IP): 400 μL of anti-FLAG M2 agarose beads (Sigma A2220) were incubated 2 h with cell lysates under gentle agitation. Then IP beads were washed 3 times in buffer B before being resuspended into 500 µL of buffer C (100 mM Tris-HCl pH=8, 100 g/L, 1% SDS, 10 mM EDTA) to reverse cross-linking for 18 h at 65°C. WCE and IP DNA fragments were recovered and purified using QIAquick PCR purification kit (Qiagen). Remaining RNA was removed by RNase A treatment followed by a second DNA purification step.
|
| Label |
Cy5
|
| Label protocol |
Labelling was done by Roche Nimblegen (Madison, WI, USA) using the standard Nimblegen protocol. ChIP DNA was labelled with Cy5 and control DNA was labelled with Cy3.
|
| |
|
| |
| Hybridization protocol |
Hybridization was done by Roche Nimblegen (Madison, WI, USA) following the standard Nimblegen protocol.
|
| Scan protocol |
Scanning was done by Roche Nimblegen (Madison, WI, USA) following the standard Nimblegen protocol.
|
| Data processing |
Identification of peaks corresponding to Zur binding sites on the Bacillus subtilis chromosome was done as described previously (Reppas et al., 2006, Mol Cell 24: 747-757 [PMID 17157257]). Signal intensities were converted as log2 ratio (ChIP-DNA/Control-DNA) and corrected for dye bias using Loess regression on the MA plot. The signal was smoothed by two rounds of sliding window averaging. The sliding window contained 29 probes (the middle probe and 14 probes on each side) corresponding to approximately 320 bp.
|
| |
|
| Submission date |
Jan 05, 2015 |
| Last update date |
Jan 06, 2015 |
| Contact name |
Sandrine Auger |
| E-mail(s) |
sandrine.auger@jouy.inra.fr
|
| Phone |
33 1 34 65 25 22
|
| Organization name |
INRA
|
| Lab |
MICALIS
|
| Street address |
INRA-Domaine de Vilvert
|
| City |
Jouy-en-Josas |
| ZIP/Postal code |
F-78350 |
| Country |
France |
| |
|
| Platform ID |
GPL13168 |
| Series (1) |
| GSE64671 |
Genome-wide identification of Bacillus subtilis Zur-binding sites. |
|
