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Sample GSM996307 Query DataSets for GSM996307
Status Public on Mar 28, 2013
Title Jurkat_ImmunoTRAP_rep2 [Agilent-014707]
Sample type genomic
 
Channel 1
Source name Jurkat cells
Organism Homo sapiens
Characteristics antibody: IgG
cell line: Jurkat cells
Treatment protocol After fixation and permeabilization, cells were washed in TS buffer (100 µM tris-HCl, 150 mM NaCl, pH 7.0). Endogenous peroxidase activity was quenched with 0.5% H2O2 in TS for 10 minutes at RT. After another TS wash, samples were blocked in 5% donkey serum in TS for 20 minutes. mAb 5E10 (a gift from R. van Driel, University of Amsterdam, The Netherlands) was used to detect PML protein. A secondary antibody conjugated with horseradish peroxidase was then added to the samples. Biotinylated tyramide (Invitrogen) was diluted in 0.1M boric acid and 0.003% H2O2 and then incubated on the samples for 2 minutes. Washing the cells three times in TS buffer stopped the reaction. After this, the samples were either processed for immunofluorescence microscopy or for affinity purification of the PML NB-associated chromatin.
Growth protocol Jurkat cells were cultured in RPMI (Invitrogen) supplemented with 10% FBS (Wisent), 10 µg/ml penicillin and streptomycin (Invitrogen), and 2 mM L-glutamine (Invitrogen).
Extracted molecule genomic DNA
Extraction protocol For affinity purification, the cells were scrapped off the slides into an eppendorf tube containing PBS. The samples were pelleted at 2900g at 4 °C for 25 minutes. The pellet was resuspended in 5M urea, 2M NaCl, and 10 nM EDTA. This suspension was then sonicated in a Misonix 3000 sonicator equipped with a Cup Horn in an iced water bath with the following program (5 sec pulse/15 sec pause repeated 15 times at power level 4). The program was repeated 4 more times with changes of the iced water bath between repetitions. The sample was then centrifuged at 13000 rpm for 15 minutes at 4 °C. The supernatant was then dialyzed overnight in PBS. Streptavidin agarose beads (Invitrogen) were washed three times in PBS and the supernatant was incubated with the beads for 3-4 hours at 4 °C. The beads were washed with the following buffers in this order: PBS, TSE150 (20 mM tris pH 8.0, 1% triton X-100, 0.1% SDS, 2 mM EDTA, 150 mM NaCl), TSE 500 (20 mM tris pH 8.0, 1% triton X-100, 0.1% SDS, 2 mM EDTA, 500 mM NaCl), and TE (10 mM tris pH 8.0, 1 mM EDTA). The beads were collected in TE buffer and the formaldehyde cross-links were reversed by shaking the samples at 65 °C overnight. The slurry was then treated with RNase A (2 µg/ml) (Fermentas) for 30 minutes at 37 °C and then with proteinase K (100 µg/ml) (Fermentas) for 6 hours at 37 °C.
Label Cy3
Label protocol The DNA was phenol extracted and ethanol precipitated using glycogen as carrier. The DNA was amplified using the Whole Genome Amplification (WGA) kit (Sigma).
 
Channel 2
Source name Jurkat cells
Organism Homo sapiens
Characteristics cell line: Jurkat cells
antibody: PML ImmunoTRAP
Treatment protocol After fixation and permeabilization, cells were washed in TS buffer (100 µM tris-HCl, 150 mM NaCl, pH 7.0). Endogenous peroxidase activity was quenched with 0.5% H2O2 in TS for 10 minutes at RT. After another TS wash, samples were blocked in 5% donkey serum in TS for 20 minutes. mAb 5E10 (a gift from R. van Driel, University of Amsterdam, The Netherlands) was used to detect PML protein. A secondary antibody conjugated with horseradish peroxidase was then added to the samples. Biotinylated tyramide (Invitrogen) was diluted in 0.1M boric acid and 0.003% H2O2 and then incubated on the samples for 2 minutes. Washing the cells three times in TS buffer stopped the reaction. After this, the samples were either processed for immunofluorescence microscopy or for affinity purification of the PML NB-associated chromatin.
Growth protocol Jurkat cells were cultured in RPMI (Invitrogen) supplemented with 10% FBS (Wisent), 10 µg/ml penicillin and streptomycin (Invitrogen), and 2 mM L-glutamine (Invitrogen).
Extracted molecule genomic DNA
Extraction protocol For affinity purification, the cells were scrapped off the slides into an eppendorf tube containing PBS. The samples were pelleted at 2900g at 4 °C for 25 minutes. The pellet was resuspended in 5M urea, 2M NaCl, and 10 nM EDTA. This suspension was then sonicated in a Misonix 3000 sonicator equipped with a Cup Horn in an iced water bath with the following program (5 sec pulse/15 sec pause repeated 15 times at power level 4). The program was repeated 4 more times with changes of the iced water bath between repetitions. The sample was then centrifuged at 13000 rpm for 15 minutes at 4 °C. The supernatant was then dialyzed overnight in PBS. Streptavidin agarose beads (Invitrogen) were washed three times in PBS and the supernatant was incubated with the beads for 3-4 hours at 4 °C. The beads were washed with the following buffers in this order: PBS, TSE150 (20 mM tris pH 8.0, 1% triton X-100, 0.1% SDS, 2 mM EDTA, 150 mM NaCl), TSE 500 (20 mM tris pH 8.0, 1% triton X-100, 0.1% SDS, 2 mM EDTA, 500 mM NaCl), and TE (10 mM tris pH 8.0, 1 mM EDTA). The beads were collected in TE buffer and the formaldehyde cross-links were reversed by shaking the samples at 65 °C overnight. The slurry was then treated with RNase A (2 µg/ml) (Fermentas) for 30 minutes at 37 °C and then with proteinase K (100 µg/ml) (Fermentas) for 6 hours at 37 °C.
Label Cy5
Label protocol The DNA was phenol extracted and ethanol precipitated using glycogen as carrier. The DNA was amplified using the Whole Genome Amplification (WGA) kit (Sigma).
 
 
Hybridization protocol A 5 µg Cy5-labeled and 5 µg of Cy3-labeled DNAs were combined in a total volume of 158 µl. The 158 µl were mixed with 50 µl of 1 mg/ml of Human Cot-1 DNA, 52 µl of 10x Agilent Blocking Agent, 260 µl of 2x Agilent Hybridization Buffer. Samples were heated at 95°C for 3 min, and then incubated at 37°C for 30 min. 490 µl of the sample were applied to the 1x244 promoter array assembled in a chamber and incubated in a rotisserie hybridization oven at 65°C and 20 r.p.m. for 40 h. After hybridization the microarray slide was washed with Oligo aCGH/ChIP-on-chip Wash Buffer for 5 min at room temperature, followed by a wash for 5 min at 31°C.
Scan protocol Agilent Feature Extraction software v9.5 was used.
Data processing Data were pre-processed using the variance-stabilizing normalization algorithm with default parameter settings, as implemented in the vsn package (v3.8.0) of the BioConductor open-source library
 
Submission date Sep 03, 2012
Last update date Mar 29, 2013
Contact name Paul C Boutros
E-mail(s) Paul.Boutros@utoronto.ca
Organization name Ontario Institute for Cancer Research
Street address 101 College Street, Suite 800
City Toronto
State/province Ontario
ZIP/Postal code M5G 0A3
Country Canada
 
Platform ID GPL4125
Series (1)
GSE40554 Identifying chromatin associations with promyelocytic leukemia nuclear bodies using immunoTRAP

Data table header descriptions
ID_REF
VALUE VSN-normalized log ratios of Cy5/Cy3

Data table
ID_REF VALUE
1 0.13613845378401
2 0.324525696960298
3 0.64120664845801
4 -0.103473358153973
5 0.794971077556593
6 2.44867617792885
7 0.336084688456513
8 0.0407295803795638
9 -0.161383907633425
10 1.48304508660054
11 0.54309210450242
12 -0.178847515487289
13 0.406477245791679
14 0.471507564572027
15 0.250196544315791
16 0.14683168392302
17 -0.0394063876088246
18 -0.221809100239401
19 0.830477220576136
20 0.0565127972024637

Total number of rows: 243485

Table truncated, full table size 5950 Kbytes.




Supplementary file Size Download File type/resource
GSM996307_June13_251470711661_S01_ChIP-v1_95_May07.txt.gz 69.3 Mb (ftp)(http) TXT
Processed data included within Sample table

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