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Sample GSM537116 Query DataSets for GSM537116
Status Public on Dec 10, 2010
Title IgG vs. C17/C20 1 of 2 100126
Sample type genomic
 
Channel 1
Source name SOM010PK
Organism Homo sapiens
Characteristics antibody: IgG
cell line: Jurkat
fraction: NONSPECIFIC-IgG
Extracted molecule genomic DNA
Extraction protocol Jurkat 1x10E6 cells were fixed with 1% formaldehyde in culture medium for 10 min at room temperature followed by quenching with 0.125 M glycine for 5 min. The cells were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH8.0) for at least 30 min on ice. The crosslinked chromatin was sheared to an average size of 500 bp by ten 30-second pulses using a BiorupterTM sonicator (Diagenode). The lysate was then centrifuged to remove cell debris, and the chromatin was quantified in a UV spectrophotometer. The chromatin (400 µg) was diluted 10-fold in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA 16.7 mM Tris pH8.10, 167 mM NaCl) and pre-cleared with 25 µl magnetic beads (in BSA) for 2 h at 4C. The chromatin supernatant was then incubated overnight with 5 µg of purified antibody at 4C. After incubation with protein A magnetic beads (50 µl) for 2h at 4C, the immune complexes were collected by centrifugation and washed with the following buffers each for 10 min at 4C; RIPA buffer (150 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), high-salt buffer (500 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS, 1% NP40) LiCl buffer (250 mM LiCl, 50 mM Tris pH8.0, 0.5% Na deoxycholate, 1% NP40) and 2x TE (20 mM Tris pH8.0, 2 mM EDTA). The protein-DNA complexes were eluted from the beads in 450 µl elution buffer (1% SDS, 100 mM NaHCO3) at 55C for 2 h followed by the addition of proteinaseK to 500 µg/ml and overnight incubaton at 65C. Genomic DNA was isolated from the precipitated material as well as from the sheared chromatin input (1/100 of the material used for ChIP) by phenol extraction and ethanol precipitation.
Label Cy3
Label protocol 4 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers (ChIP DNA isolated from normal rabbit IgG-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
 
Channel 2
Source name SOM010PG
Organism Homo sapiens
Characteristics antibody: ERG C17/20
antibody manufacturer: Santa Cruz Biotechnolgy
cell line: Jurkat
fraction: EXPERIMENTAL
Extracted molecule genomic DNA
Extraction protocol Jurkat 1x10E6 cells were fixed with 1% formaldehyde in culture medium for 10 min at room temperature followed by quenching with 0.125 M glycine for 5 min. The cells were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH8.0) for at least 30 min on ice. The crosslinked chromatin was sheared to an average size of 500 bp by ten 30-second pulses using a BiorupterTM sonicator (Diagenode). The lysate was then centrifuged to remove cell debris, and the chromatin was quantified in a UV spectrophotometer. The chromatin (400 µg) was diluted 10-fold in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA 16.7 mM Tris pH8.10, 167 mM NaCl) and pre-cleared with 25 µl magnetic beads (in BSA) for 2 h at 4C. The chromatin supernatant was then incubated overnight with 5 µg of purified antibody at 4C. After incubation with protein A magnetic beads (50 µl) for 2h at 4C, the immune complexes were collected by centrifugation and washed with the following buffers each for 10 min at 4C; RIPA buffer (150 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), high-salt buffer (500 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS, 1% NP40) LiCl buffer (250 mM LiCl, 50 mM Tris pH8.0, 0.5% Na deoxycholate, 1% NP40) and 2x TE (20 mM Tris pH8.0, 2 mM EDTA). The protein-DNA complexes were eluted from the beads in 450 µl elution buffer (1% SDS, 100 mM NaHCO3) at 55C for 2 h followed by the addition of proteinaseK to 500 µg/ml and overnight incubaton at 65C. Genomic DNA was isolated from the precipitated material as well as from the sheared chromatin input (1/100 of the material used for ChIP) by phenol extraction and ethanol precipitation.
Label Cy5
Label protocol 4 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers (ChIP DNA isolated from normal rabbit IgG-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
 
 
Hybridization protocol The labeled ChIP DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in 45 ul of buffer containing 20% formamide, 1.2 M betaine, 0.1 ug/ul herring sperm DNA and 10 ug of human COT1 DNA (Invitrogen). Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C, and then washed in 42C 0.2% SDS/0.2x SSC, room temperature 0.2x SSC, and 0.05x SSC. Hybridization buffers and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html)
Scan protocol Arrays were scanned on an Axon 4000B scanner per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
Data processing Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction. Roche NimbleGen scales the GFF files by subtracting the bi-weight mean for the log-ratio values from each log-ratio value.
Procedure for Lowess_ratios annalysis
* take pair data from Nimblegen
* normalize them with limma package (script available from Metzler hompage
(http://watson.nci.nih.gov/twiki/bin/view/Main/MeltzerLabSoftware)
* create file with required informations for ACME sofware/Analysis (like ratio (R/G) chromosomal position etc (same as for the first analysis already done)
run program (parameters: window size 2000 bp, threshold 0,90)
calculate p-values (FDR-analysis; false discovery rate) create list with: chromosome, chromosomal position, p-value, accession number
 
Submission date Apr 23, 2010
Last update date Dec 10, 2010
Contact name Liliana H. Mochmann
E-mail(s) liliana.mochmann@charite.de
Phone 4903084454575
Fax 4903084454468
Organization name Charite
Department Hematology and Oncology
Lab AG BALDUS
Street address Hindenburgdamm 30
City Berlin
State/province Berlin
ZIP/Postal code 12203
Country Germany
 
Platform ID GPL6325
Series (1)
GSE21495 Genome wide screen reveals WNT11, a noncanonical WNT gene, as a target of ETS transcription factor ERG

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

Data table
ID_REF VALUE
CHR10P001020948 -0.14
CHR10P001021048 0.48
CHR10P001026162 -0.73
CHR10P001022248 -0.22
CHR10P001023448 0.25
CHR10P001023148 -0.47
CHR10P001026962 -0.39
CHR10P001021248 -0.27
CHR10P001021748 -0.06
CHR10P001024348 0.63
CHR10P001026062 -0.90
CHR10P001021648 0.32
CHR10P001023548 -0.19
CHR10P001023248 -0.11
CHR10P001027262 -0.37
CHR10P001022548 -0.22
CHR10P001025462 -0.06
CHR10P001026762 0.32
CHR10P001027562 -0.28
CHR10P001021348 -0.14

Total number of rows: 378943

Table truncated, full table size 7959 Kbytes.




Supplementary file Size Download File type/resource
GSM537116_100126_532_pair.txt.gz 7.0 Mb (ftp)(http) TXT
GSM537116_100126_635_pair.txt.gz 6.9 Mb (ftp)(http) TXT
GSM537116_IgG_vs._C17C20_1of2_100126_norm_ratios.gff.gz 4.5 Mb (ftp)(http) GFF
GSM537116_sig100126_Lowess_ratios.gff.gz 55.0 Kb (ftp)(http) GFF
Processed data included within Sample table
Processed data provided as supplementary file
Processed data are available on Series record

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