|
| Status |
Public on Jul 01, 2010 |
| Title |
Sigma70_rep_3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Sigma70 ChIP DNA
|
| Organism |
Cereibacter sphaeroides 2.4.1 |
| Characteristics |
strain: Wild-type
antibody: Sigma70
|
| Growth protocol |
cells were cultured in minimal succinate based medium under anaerobic photosynthetic conditions until mid-exponential growth phase
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
cells were fixed with 1% formaldehyde in culture medium for 5 minutes at 30°C followed by quenching with 0.125 M clycine for 30 minutes on ice. The cells were washed twice with ice cold PBS, frozen in dry/ethanol bath and stored at -80°C. Cells were resuspended in 500uL IP buffer (100 mM Tris pH 8, 300 mM NaCl, 2% TritinX-100), and sonicated (50% output, level 6) for 20 seconds 8 times to shear the DNA to fragments of 1 kbp in average. 50 units of micrococcal DNase and 0.5ug of RNaseA were added to the lysate and incubated for 1 hour at 4°C to reduce fragment size to 500 bp in avergage and degrade RNA. To stop the nucleases EDTA was added to 10mM final concentration. The lysate was cenrtrifuged to remove cell debris and 100 uL was saved for Input DNA. The lysate was was incubated with polyclonal antibodies against RpoE or Beta' at 4°C over night. Then, ProteinA coated sepharose beads were added to the lysate, which was incubated for another 3 hours to capture antibodies. Beads were washed once with LiCl buffer (125 mM LiCL, 50 mM Tris pH 8, 1 % TritonX-100), twice with 600 mM NaCl Tris buffer, twice with 300mM NaCl Tris buffer and twive with TE buffer (10 mM Tris ph 8, 1 mM EDTA). Beads were ressupended in 200 uL Elution buffer (50 mM Tris pH 8, 10 nM EDTA, 1% SDS) and incubated at 65°C for 15 hours to reverse cross linking. DNA was cleaned using QIAGen DNA purification kit.
|
| Label |
Cy5
|
| Label protocol |
Before labelling, the ChIP DNA was amplified using ligation mediated PCR. 1 ug of DNA was labelled using the NimbleGen Dual-color DNA labelling Kit (05223547001) according to the manufacturer's protocol.
|
| |
|
| Channel 2 |
| Source name |
Input DNA
|
| Organism |
Cereibacter sphaeroides 2.4.1 |
| Characteristics |
strain: Wild-type
antibody: none
|
| Growth protocol |
cells were cultured in minimal succinate based medium under anaerobic photosynthetic conditions until mid-exponential growth phase
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
cells were fixed with 1% formaldehyde in culture medium for 5 minutes at 30°C followed by quenching with 0.125 M clycine for 30 minutes on ice. The cells were washed twice with ice cold PBS, frozen in dry/ethanol bath and stored at -80°C. Cells were resuspended in 500uL IP buffer (100 mM Tris pH 8, 300 mM NaCl, 2% TritinX-100), and sonicated (50% output, level 6) for 20 seconds 8 times to shear the DNA to fragments of 1 kbp in average. 50 units of micrococcal DNase and 0.5ug of RNaseA were added to the lysate and incubated for 1 hour at 4°C to reduce fragment size to 500 bp in avergage and degrade RNA. To stop the nucleases EDTA was added to 10mM final concentration. The lysate was cenrtrifuged to remove cell debris and 100 uL was saved for Input DNA. The lysate was was incubated with polyclonal antibodies against RpoE or Beta' at 4°C over night. Then, ProteinA coated sepharose beads were added to the lysate, which was incubated for another 3 hours to capture antibodies. Beads were washed once with LiCl buffer (125 mM LiCL, 50 mM Tris pH 8, 1 % TritonX-100), twice with 600 mM NaCl Tris buffer, twice with 300mM NaCl Tris buffer and twive with TE buffer (10 mM Tris ph 8, 1 mM EDTA). Beads were ressupended in 200 uL Elution buffer (50 mM Tris pH 8, 10 nM EDTA, 1% SDS) and incubated at 65°C for 15 hours to reverse cross linking. DNA was cleaned using QIAGen DNA purification kit.
|
| Label |
Cy3
|
| Label protocol |
Before labelling, the ChIP DNA was amplified using ligation mediated PCR. 1 ug of DNA was labelled using the NimbleGen Dual-color DNA labelling Kit (05223547001) according to the manufacturer's protocol.
|
| |
|
| |
| Hybridization protocol |
4 ug of labelled DNA were hybridized on 385K NimbleGen custom arrays according to NimbleGen's protocol.
|
| Scan protocol |
Arrays were scanned on an Axon 4000B scanner according to the manufacturer's protocol.
|
| Description |
ChIP-chip sigma70 in Wild-type cells
|
| Data processing |
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction. The signal from Beta' ChIP was subtracted from the signals from FnrL and Sigma70 ChIP using loess normalization to remove background signal.
|
| |
|
| Submission date |
May 27, 2010 |
| Last update date |
May 28, 2010 |
| Contact name |
Yann S Dufour |
| Organization name |
University of Wisconsin - Madison
|
| Department |
Bacteriology
|
| Lab |
Timothy Donohue
|
| Street address |
1550 Linden Drive
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
| |
|
| Platform ID |
GPL10463 |
| Series (1) |
| GSE22027 |
FnrL regulon in Rhodobacter sphaeroides |
|
