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| Status |
Public on Jan 26, 2015 |
| Title |
Bsub_LSB003_LPDM_T2_rep2 |
| Sample type |
genomic |
| |
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| Channel 1 |
| Source name |
ChIP_DNA from Bsub strain expressing WalR-SPA protein, T2 phase
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: LSB003
growth phase: T2
chip antibody: mouse anti-FLAG M2 (Sigma)
|
| Growth protocol |
Cells of strain LSB003 were grown in LPDM with the addition of 1 µg ml-1 erythromycin plus 25 µg ml-1 lincomycin (MLS) and 100 mM IPTG with vigorous shaking at 37oC. Two hours after the onset of phosphate starvation state (T2), 50 ml were treated with formaldehyde.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Crosslinking was performed by the addition of formaldehyde to a final concentration of 1% followed by 20 min incubation at room temperature with slow shaking. The cultures were then quenched with Glycine (0.36M final concentration) for 5 min at room temperature. Cells were collected by centrifugation at 4oC and the pellets were washed twice with 50 ml ice-cold Buffer A (10 mM Tris-HCl pH 7.5, 150 mM NaCl) before being snap frozen in a dry ice / ethanol bath. Pellets were thawed at 37oC and resuspended in 0.5ml buffer B (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2 mM EDTA, 0.1% Triton X-100) supplemented with 3 mg ml-1 lysozyme and 0.1 mg ml-1 RNaseA (final concentrations) and incubated for 30 min at 37oC, then the sample volume was adjusted to 1 ml with buffer B and samples placed on ice for 10 min. Genomic DNA was sheared by sonication (Diagenode Bioruptor Twin sonicator) into fragments between 0.2 and 1 kb. Sonicated samples were centrifuged for 10 min at 20 000 g (4oC) to remove insoluble debris. Fifty microliteres of the cleared lysates was removed to serve as the CONTROL sample, to which 50 microliters of 10X buffer C (10 mM Tris-HCl pH 8, 10% SDS, 10 mM EDTA) and 400 microliters buffer B were added and the sample was stored on ice. The remaining cleared lysates were each added to 40 microliters anti-FLAG M2-agarose resin (Sigma-Aldrich; A2220) equilibrated with ice-cold buffer B (according to manufacturers’ instructions) and incubated for two hours at 4oC with mild agitation. Beads were then collected by centrifugation for 1 min at 5000 g (4oC) and washed three times with 0.5 ml ice-cold buffer B. To reverse the crosslinking beads were transferred to new 1.5 ml eppendorf tubes, resuspended in 0.5 ml 1X buffer C and incubated along with the CONTROL samples for ~20 hours at 65oC with shaking (950 rpm). ChIP samples were collected by centrifugation for 1 min at 13 300 rpm (4oC). DNA from both ChIP and CONTROL samples was purified using the QIAquick PCR purification kit (Qiagen, 28106), eluted in 50 microliters H2O and treated with 0.02 mg ml-1 RNaseA (Roche, 11119915001) for 30 min at 37oC. RNase was eliminated using the QIAquick PCR purification kit and samples were eluted in 30 microliters H2O. Equal concentrations of ChIP DNA and CONTROL samples were amplified using the GenomePlex complete Whole Genome Amplification (WGA) kit (Sigma-Aldrich; WGA2-50RXN) as per manufacturers’ instructions and purified using the QIAquick PCR purification kit and eluted in 30 microliters H2O. Ten microliters of each of the purified amplified samples was subjected to a second round of amplification using the WGA kit as per manufacturer’s instructions, purified using the QIAquick PCR purification kit and eluted in 30 microliters H2O. 3.5-4 micrograms of twice-amplified ChIP DNA and CONTROL samples (of both tagged and mock precipitation experiments) at a concentration of ~250 ng per microliter (A260/A280 ≥1.7 and A260/A230 ≥ 1.6) from three separate ChIP experiments were submitted TO NimbleGen. Crosslinking was performed by the addition of formaldehyde to a final concentration of 1% followed by 20 min incubation at room temperature with slow shaking. The cultures were then quenched with Glycine (0.36M final concentration) for 5 min at room temperature. Cells were collected by centrifugation at 4oC and the pellets were washed twice with 50 ml ice-cold Buffer A (10 mM Tris-HCl pH 7.5, 150 mM NaCl) before being snap frozen in a dry ice / ethanol bath. Pellets were thawed at 37oC and resuspended in 0.5ml buffer B (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2 mM EDTA, 0.1% Triton X-100) supplemented with 3 mg ml-1 lysozyme and 0.1 mg ml-1 RNaseA (final concentrations) and incubated for 30 min at 37oC, then the sample volume was adjusted to 1 ml with buffer B and samples placed on ice for 10 min. Genomic DNA was sheared by sonication (Diagenode Bioruptor Twin sonicator) into fragments between 0.2 and 1 kb. Sonicated samples were centrifuged for 10 min at 20 000 g (4oC) to remove insoluble debris. Fifty microliteres of the cleared lysates was removed to serve as the CONTROL sample, to which 50 l of 10X buffer C (10 mM Tris-HCl pH 8, 10% SDS, 10 mM EDTA) and 400 l buffer B were added and the sample was stored on ice. The remaining cleared lysates were each added to 40 l anti-FLAG M2-agarose resin (Sigma-Aldrich; A2220) equilibrated with ice-cold buffer B (according to manufacturers’ instructions) and incubated for two hours at 4oC with mild agitation. Beads were then collected by centrifugation for 1 min at 5000 g (4oC) and washed three times with 0.5 ml ice-cold buffer B. To reverse the crosslinking beads were transferred to new 1.5 ml eppendorf tubes, resuspended in 0.5 ml 1X buffer C and incubated along with the CONTROL samples for ~20 hours at 65oC with shaking (950 rpm). ChIP samples were collected by centrifugation for 1 min at 13 300 rpm (4oC). DNA from both ChIP and CONTROL samples was purified using the QIAquick PCR purification kit (Qiagen, 28106), eluted in 50 l H2O and treated with 0.02 mg ml-1 RNaseA (Roche, 11119915001) for 30 min at 37oC. RNase was eliminated using the QIAquick PCR purification kit and samples were eluted in 30 l H2O. Equal concentrations of ChIP DNA and CONTROL samples were amplified using the GenomePlex complete Whole Genome Amplification (WGA) kit (Sigma-Aldrich; WGA2-50RXN) as per manufacturers’ instructions and purified using the QIAquick PCR purification kit and eluted in 30 l H2O. Ten microliters of each of the purified amplified samples was subjected to a second round of amplification using the WGA kit as per manufacturer’s instructions, purified using the QIAquick PCR purification kit and eluted in 30 l H2O. 3.5-4 micrograms of twice-amplified ChIP DNA and CONTROL samples (of both tagged and mock precipitation experiments) at a concentration of ~250 ng l-1 (A260/A280 ≥1.7 and A260/A230 ≥ 1.6) from three separate ChIP experiments were submitted TO NimbleGen.
|
| Label |
Cy5
|
| Label protocol |
Labeling was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol.ChIP DNA is labelled with Cy5 and CONTROL DNA with Cy3. See www.nimblegen.com.
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| |
|
| Channel 2 |
| Source name |
Control_DNA from Bsub strain expressing WalR-SPA protein, T2 phase
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: LSB003
growth phase: T2
chip antibody: none
|
| Growth protocol |
Cells of strain LSB003 were grown in LPDM with the addition of 1 µg ml-1 erythromycin plus 25 µg ml-1 lincomycin (MLS) and 100 mM IPTG with vigorous shaking at 37oC. Two hours after the onset of phosphate starvation state (T2), 50 ml were treated with formaldehyde.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Crosslinking was performed by the addition of formaldehyde to a final concentration of 1% followed by 20 min incubation at room temperature with slow shaking. The cultures were then quenched with Glycine (0.36M final concentration) for 5 min at room temperature. Cells were collected by centrifugation at 4oC and the pellets were washed twice with 50 ml ice-cold Buffer A (10 mM Tris-HCl pH 7.5, 150 mM NaCl) before being snap frozen in a dry ice / ethanol bath. Pellets were thawed at 37oC and resuspended in 0.5ml buffer B (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2 mM EDTA, 0.1% Triton X-100) supplemented with 3 mg ml-1 lysozyme and 0.1 mg ml-1 RNaseA (final concentrations) and incubated for 30 min at 37oC, then the sample volume was adjusted to 1 ml with buffer B and samples placed on ice for 10 min. Genomic DNA was sheared by sonication (Diagenode Bioruptor Twin sonicator) into fragments between 0.2 and 1 kb. Sonicated samples were centrifuged for 10 min at 20 000 g (4oC) to remove insoluble debris. Fifty microliteres of the cleared lysates was removed to serve as the CONTROL sample, to which 50 microliters of 10X buffer C (10 mM Tris-HCl pH 8, 10% SDS, 10 mM EDTA) and 400 microliters buffer B were added and the sample was stored on ice. The remaining cleared lysates were each added to 40 microliters anti-FLAG M2-agarose resin (Sigma-Aldrich; A2220) equilibrated with ice-cold buffer B (according to manufacturers’ instructions) and incubated for two hours at 4oC with mild agitation. Beads were then collected by centrifugation for 1 min at 5000 g (4oC) and washed three times with 0.5 ml ice-cold buffer B. To reverse the crosslinking beads were transferred to new 1.5 ml eppendorf tubes, resuspended in 0.5 ml 1X buffer C and incubated along with the CONTROL samples for ~20 hours at 65oC with shaking (950 rpm). ChIP samples were collected by centrifugation for 1 min at 13 300 rpm (4oC). DNA from both ChIP and CONTROL samples was purified using the QIAquick PCR purification kit (Qiagen, 28106), eluted in 50 microliters H2O and treated with 0.02 mg ml-1 RNaseA (Roche, 11119915001) for 30 min at 37oC. RNase was eliminated using the QIAquick PCR purification kit and samples were eluted in 30 microliters H2O. Equal concentrations of ChIP DNA and CONTROL samples were amplified using the GenomePlex complete Whole Genome Amplification (WGA) kit (Sigma-Aldrich; WGA2-50RXN) as per manufacturers’ instructions and purified using the QIAquick PCR purification kit and eluted in 30 microliters H2O. Ten microliters of each of the purified amplified samples was subjected to a second round of amplification using the WGA kit as per manufacturer’s instructions, purified using the QIAquick PCR purification kit and eluted in 30 microliters H2O. 3.5-4 micrograms of twice-amplified ChIP DNA and CONTROL samples (of both tagged and mock precipitation experiments) at a concentration of ~250 ng per microliter (A260/A280 ≥1.7 and A260/A230 ≥ 1.6) from three separate ChIP experiments were submitted TO NimbleGen. Crosslinking was performed by the addition of formaldehyde to a final concentration of 1% followed by 20 min incubation at room temperature with slow shaking. The cultures were then quenched with Glycine (0.36M final concentration) for 5 min at room temperature. Cells were collected by centrifugation at 4oC and the pellets were washed twice with 50 ml ice-cold Buffer A (10 mM Tris-HCl pH 7.5, 150 mM NaCl) before being snap frozen in a dry ice / ethanol bath. Pellets were thawed at 37oC and resuspended in 0.5ml buffer B (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2 mM EDTA, 0.1% Triton X-100) supplemented with 3 mg ml-1 lysozyme and 0.1 mg ml-1 RNaseA (final concentrations) and incubated for 30 min at 37oC, then the sample volume was adjusted to 1 ml with buffer B and samples placed on ice for 10 min. Genomic DNA was sheared by sonication (Diagenode Bioruptor Twin sonicator) into fragments between 0.2 and 1 kb. Sonicated samples were centrifuged for 10 min at 20 000 g (4oC) to remove insoluble debris. Fifty microliteres of the cleared lysates was removed to serve as the CONTROL sample, to which 50 l of 10X buffer C (10 mM Tris-HCl pH 8, 10% SDS, 10 mM EDTA) and 400 l buffer B were added and the sample was stored on ice. The remaining cleared lysates were each added to 40 l anti-FLAG M2-agarose resin (Sigma-Aldrich; A2220) equilibrated with ice-cold buffer B (according to manufacturers’ instructions) and incubated for two hours at 4oC with mild agitation. Beads were then collected by centrifugation for 1 min at 5000 g (4oC) and washed three times with 0.5 ml ice-cold buffer B. To reverse the crosslinking beads were transferred to new 1.5 ml eppendorf tubes, resuspended in 0.5 ml 1X buffer C and incubated along with the CONTROL samples for ~20 hours at 65oC with shaking (950 rpm). ChIP samples were collected by centrifugation for 1 min at 13 300 rpm (4oC). DNA from both ChIP and CONTROL samples was purified using the QIAquick PCR purification kit (Qiagen, 28106), eluted in 50 l H2O and treated with 0.02 mg ml-1 RNaseA (Roche, 11119915001) for 30 min at 37oC. RNase was eliminated using the QIAquick PCR purification kit and samples were eluted in 30 l H2O. Equal concentrations of ChIP DNA and CONTROL samples were amplified using the GenomePlex complete Whole Genome Amplification (WGA) kit (Sigma-Aldrich; WGA2-50RXN) as per manufacturers’ instructions and purified using the QIAquick PCR purification kit and eluted in 30 l H2O. Ten microliters of each of the purified amplified samples was subjected to a second round of amplification using the WGA kit as per manufacturer’s instructions, purified using the QIAquick PCR purification kit and eluted in 30 l H2O. 3.5-4 micrograms of twice-amplified ChIP DNA and CONTROL samples (of both tagged and mock precipitation experiments) at a concentration of ~250 ng l-1 (A260/A280 ≥1.7 and A260/A230 ≥ 1.6) from three separate ChIP experiments were submitted TO NimbleGen.
|
| Label |
Cy3
|
| Label protocol |
Labeling was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol.ChIP DNA is labelled with Cy5 and CONTROL DNA with Cy3. See www.nimblegen.com.
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed by NimbleGen Systems Inc., Madison, WI, USA following their standard operating protocol. See www.nimblegen.com.
|
| Scan protocol |
Scanning was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
|
| Data processing |
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction. Data files were loaded into Bioconductor and preprocessed through Ringo, using the 'nimblegen' method. Combination of replicates and smoothing was accomplished through Ringo's 'computeRuningMedians' function, using a winHalfSize of 350.
merged and smoothed log2 (ChIP/Input) ratio
|
| |
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| Submission date |
Jan 26, 2015 |
| Last update date |
Jan 26, 2015 |
| Contact name |
Karsten Hokamp |
| E-mail(s) |
kahokamp@tcd.ie
|
| Phone |
+35318962719
|
| Organization name |
Trinity College Dublin
|
| Department |
Smurfit Institute of Genetics
|
| Street address |
College Green
|
| City |
Dublin |
| ZIP/Postal code |
2 |
| Country |
Ireland |
| |
|
| Platform ID |
GPL13168 |
| Series (1) |
| GSE65272 |
Bacillus subtilis WalR (YycF) ChIP-chip Bacillus subtilis; Bacillus subtilis subsp. subtilis str. 168 |
|
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1591528_41984402_532.v4.pair.gz |
8.1 Mb |
(ftp)(http) |
PAIR |
| GSM1591528_41984402_635.v4.pair.gz |
8.0 Mb |
(ftp)(http) |
PAIR |
| Processed data are available on Series record |
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