|
| Status |
Public on Aug 03, 2012 |
| Title |
RpoHII replicate 2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
RpoHII ChIP DNA
|
| Organism |
Cereibacter sphaeroides 2.4.1 |
| Characteristics |
strain: RSP_0601 deletion mutant complemented with a plasmid expressing wild type RSP_0601 under the control of a lac promoter.
antibody: custom rabbit polyclonal antibody against RSP_0601.
|
| Growth protocol |
Cells were cultured in minimal succinate based medium under aerobic conditions at 30C until early-exponential growth phase when 100 uM IPTG was added to induce the expression of RpoHII for 3 hours before harvesting cells.
|
| 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 RpoHII 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: RSP_0601 deletion mutant complemented with a plasmid expressing wild type RSP_0601 under the control of a lac promoter.
antibody: none.
|
| Growth protocol |
Cells were cultured in minimal succinate based medium under aerobic conditions at 30C until early-exponential growth phase when 100 uM IPTG was added to induce the expression of RpoHII for 3 hours before harvesting cells.
|
| 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 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.
|
| Data processing |
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction. The biological replicates were normalized to each others using quantile normalization. The data was smoothed using a moving median window of 100 bp.
|
| |
|
| Submission date |
Jul 29, 2012 |
| Last update date |
Aug 03, 2012 |
| 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 (2) |
| GSE39711 |
RpoHI and RpoHII regulons in Rhodobacter sphaeroides 2.4.1 from chromatin immuno-precipitation |
| GSE39806 |
RpoHI and RpoHII regulons in Rhodobacter sphaeroides 2.4.1 |
|
