|
| Status |
Public on Jan 01, 2012 |
| Title |
SigA2stat_29851002 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
ChIP_DNA from BSB1 strain expressing SigA-SPA protein, stationary phase
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: BSB1
growth phase: stationary
chip antibody: mouse anti-FLAG M2
antibody vendor: Sigma
antibody catalog number: A2220
antibody lot number: 016K6290
|
| Growth protocol |
BSB1 cells expressing the SigA-SPA protein were grown in 2 liters of LB medium (Erythromycin 0.6 mg/L, IPTG 0.5mM) supplemented with 0.3% glucose with vigorous shaking at 37°C. At stationary growth phase (OD600nm ≈ 1.5), 400mL was treated with formaldehyde.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were treated with formaldehyde 0.6 % final concentration, and cross-linking was carried out for 20 minutes at room temperature before quenching with 125 mM glycine (final concentration). Cells were harvested, washed twice with buffer A (10 mM Tris-HCl pH 7.5, 150 mM NaCl) and treated with lysozyme (1 mg/mL) in 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 of RNase A. DNA was then sheared by sonication in order to obtain 0.2 to 1 Kbp fragments. Cell debris were removed by centrifugation at 20’000g for 30 minutes. 400 μL of Anti-FLAG M2 agarose beads (Sigma) washed with buffer B were added to the supernatant and incubation was continued for 2 h with rotation at 4 °C. Beads were then harvested, washed 3 times with large volumes of buffer B and resuspended in 500 μL of buffer C (100 mM Tris-HCl pH 8, 100 g/L SDS, 10 mM EDTA). Cross-links were dissociated by incubation at 65 °C for 18 hours. DNA fragments co-purified with SigA-SPA were extracted using the QIAquick PCR Purification kit (Qiagen). Contaminating RNA was treated with RNase A and a second purification of the DNA fragments was done. These fragments (ChIP-DNA) and DNA isolated from the whole cell extract fraction before the immunopurification (control DNA) were sent to NimbleGen.
|
| Label |
Cy5
|
| Label protocol |
Standard NimbleGen protocol. ChIP DNA is labelled with Cy5 and control DNA is labelled with Cy3.
|
| |
|
| Channel 2 |
| Source name |
Control_DNA from BSB1 strain expressing SigA-SPA protein, stationary phase
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: BSB1
growth phase: stationary
chip antibody: none
|
| Growth protocol |
BSB1 cells expressing the SigA-SPA protein were grown in 2 liters of LB medium (Erythromycin 0.6 mg/L, IPTG 0.5mM) supplemented with 0.3% glucose with vigorous shaking at 37°C. At stationary growth phase (OD600nm ≈ 1.5), 400mL was treated with formaldehyde.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were treated with formaldehyde 0.6 % final concentration, and cross-linking was carried out for 20 minutes at room temperature before quenching with 125 mM glycine (final concentration). Cells were harvested, washed twice with buffer A (10 mM Tris-HCl pH 7.5, 150 mM NaCl) and treated with lysozyme (1 mg/mL) in 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 of RNase A. DNA was then sheared by sonication in order to obtain 0.2 to 1 Kbp fragments. Cell debris were removed by centrifugation at 20’000g for 30 minutes. 400 μL of Anti-FLAG M2 agarose beads (Sigma) washed with buffer B were added to the supernatant and incubation was continued for 2 h with rotation at 4 °C. Beads were then harvested, washed 3 times with large volumes of buffer B and resuspended in 500 μL of buffer C (100 mM Tris-HCl pH 8, 100 g/L SDS, 10 mM EDTA). Cross-links were dissociated by incubation at 65 °C for 18 hours. DNA fragments co-purified with SigA-SPA were extracted using the QIAquick PCR Purification kit (Qiagen). Contaminating RNA was treated with RNase A and a second purification of the DNA fragments was done. These fragments (ChIP-DNA) and DNA isolated from the whole cell extract fraction before the immunopurification (control DNA) were sent to NimbleGen.
|
| Label |
Cy3
|
| Label protocol |
Standard NimbleGen protocol. ChIP DNA is labelled with Cy5 and control DNA is labelled with Cy3.
|
| |
|
| |
| Hybridization protocol |
Standard NimbleGen protocol.
|
| Scan protocol |
Standard NimbleGen protocol.
|
| Description |
Stationary phase, replicate 2.
|
| Data processing |
Identification of peaks corresponding to SigA 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 |
Mar 03, 2011 |
| Last update date |
Jan 01, 2012 |
| Contact name |
Nathalie Pigeonneau |
| E-mail(s) |
nathalie.pigeonneau@jouy.inra.fr
|
| Organization name |
INRA
|
| Department |
Micalis
|
| Lab |
IFPC_Ph. Noirot
|
| Street address |
domaine de Vilvert
|
| City |
Jouy en Josas |
| ZIP/Postal code |
78 352 |
| Country |
France |
| |
|
| Platform ID |
GPL13168 |
| Series (3) |
| GSE27650 |
Bacillus subtilis SigA ChIP-chip (BsubT1 array) |
| GSE27652 |
Bacillus subtilis SigA ChIP-chip |
| GSE27665 |
Bacillus subtilis SigA ChIP-chip (BsubT2 array) |
|
