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| Status |
Public on Oct 11, 2006 |
| Title |
PolIIa_rep3 |
| Sample type |
genomic |
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| Source name |
HeLa S3 PolIIa IP
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| Organism |
Homo sapiens |
| Characteristics |
HeLa S3 PolIIa IP
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| Extracted molecule |
genomic DNA |
| Label |
biotin
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| Description |
DNA was isolated from chromatin immunoprecipitation. Briefly, HeLa S3 cells were first crosslinked with dimethyl adipimidate (DMA) (Pierce) for 10 min, washed with PBS and then crosslinked with formaldehyde for 10 min. Briefly, 8WG16 (Covance) and 4H8 (AbCam) antibodies wereincubated with a 50:50 mix of Dynal protein A/G beads for more than 16 h at 4°C in PBS with 5 mg/ml BSA. After washing in PBS, beads with bound antibody were incubated with chromatin from approximately 2 × 107 cells for more than 16 h at 4°C. Beads were washed eight times with RIPA buffer (50mM HEPES pH 7.6, 1 mM EDTA, 0.7% DOC, 1% IGEPAL, 0.5M LiCl) before DNA was eluted at 65°C in TE/1% SDS. Crosslinks were reversed by incubating at 65°C for more than 12 h followed by proteinase K treatment, phenol extraction and RNase treatment. Isolated DNA was then amplified isothermally using random nonamer primers and Klenow polymerase (Invitrogen) for more than 4 h, yielding approximately 2 ?g of DNA per IP. DNA was prepared and hybridized on Affymetrix ENCODE oligonucleotide tiled arrays using the fragmentation, hybridization, staining and scanning procedure described by Kennedy et al. [24]. Affymetrix ENCODE microarrays have interrogating 25mer oligonucleotide probes tiled every 20 bp on average. A sample of chromatin was set aside before IP and used to represent the input DNA.
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| Data processing |
To take into account probe-to-probe variability we used a generalization of the Wilcoxon signed-rank test for blocked data to generate p-values. All input and IP normalized, sign(PM-MM)max(1,|PMMM|) intensities (where PM are perfect match and MM are mismatched probes) interrogating the same chromosomal location were assigned to the same block. Probes were mapped to the genomic coordinates to ensure that no probe mapped to more than one location in any 1,000-bp window and that no two probes map to the same genomic location.
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| Submission date |
Oct 11, 2006 |
| Last update date |
Mar 09, 2012 |
| Contact name |
Alexander Brodsky |
| E-mail(s) |
alex_brodsky@brown.edu
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| Phone |
401-444-1649
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| Organization name |
Rhode Island Hospital/Brown University
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| Department |
MCB
|
| Lab |
Brodsky lab
|
| Street address |
593 Eddy St.
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| City |
PROVIDENCE |
| State/province |
RI |
| ZIP/Postal code |
02903 |
| Country |
USA |
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| Platform ID |
GPL2138 |
| Series (1) |
| GSE2735 |
ENCODE: two phosphorylation states of RNAP II ChIP-chip from HeLa cells |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM139720.CEL.gz |
9.5 Mb |
(ftp)(http) |
CEL |
| Processed data not provided for this record |
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