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Sample GSM547637 Query DataSets for GSM547637
Status Public on Jul 01, 2010
Title Beta'_rep 2
Sample type genomic
 
Channel 1
Source name Beta' ChIP DNA
Organism Cereibacter sphaeroides 2.4.1
Characteristics strain: Wild-type
antibody: Beta
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 Beta' 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

Data table header descriptions
ID_REF
VALUE scaled, log2 (ChIP/Input) ratio

Data table
ID_REF VALUE
RSPH241_F_00000001 1.776901472
RSPH241_R_00000002 1.380476833
RSPH241_F_00000003 1.695913385
RSPH241_R_00000004 1.226252467
RSPH241_F_00000005 2.001936376
RSPH241_R_00000006 1.017871929
RSPH241_F_00000007 1.777154851
RSPH241_R_00000008 1.809750951
RSPH241_F_00000009 1.610800818
RSPH241_R_00000010 1.345140252
RSPH241_F_00000011 1.449714285
RSPH241_R_00000012 1.189115904
RSPH241_F_00000013 1.346574695
RSPH241_R_00000014 1.255737684
RSPH241_F_00000015 1.708002422
RSPH241_R_00000016 1.416402683
RSPH241_F_00000017 1.768329678
RSPH241_R_00000018 1.276454186
RSPH241_F_00000019 1.307239305
RSPH241_R_00000020 1.38880654

Total number of rows: 353081

Table truncated, full table size 10841 Kbytes.




Supplementary file Size Download File type/resource
GSM547637_10544902_532.pair.gz 6.2 Mb (ftp)(http) PAIR
GSM547637_10544902_635.pair.gz 6.2 Mb (ftp)(http) PAIR
GSM547637_Betap_10544902.gff.gz 5.5 Mb (ftp)(http) GFF
Processed data included within Sample table
Processed data provided as supplementary file

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