|
| Status |
Public on Sep 29, 2007 |
| Title |
Human Ntera2 cells, Kap1 WG Array, 73429_ratio.gff |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Ntera2 cells
|
| Organism |
Homo sapiens |
| Characteristics |
KAP1 antibody immunoprecipitated DNA
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Performed following the 'Chromatin Immunoprecipitation Assay (ChIPs)' protocol provided at http://genomics.ucdavis.edu/farnham
|
| Label |
Cy5
|
| Label protocol |
The labeling of DNA samples for ChIP-ChIP analysis was performed by NimbleGen Systems, Inc. Briefly, each DNA sample (1 ug) was denatured in the presence of 5'-Cy3- or Cy5-labeled random nonamers (TriLink Biotechnologies) and incubated with 100 units (exo-) Klenow fragment (NEB) and dNTP mix [6 mM each in TE buffer (10 mM Tris/1 mM EDTA, pH 7.4; Invitrogen)] for 2 h at 37°C. Reactions were terminated by addition of 0.5 M EDTA (pH 8.0), precipitated with isopropanol, and resuspended in water.
|
| |
|
| Channel 2 |
| Source name |
Ntera2 cells
|
| Organism |
Homo sapiens |
| Characteristics |
control total input
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
see extract protocol for channel 1
|
| Label |
Cy3
|
| Label protocol |
see labeling protocol for channel 1
|
| |
|
| |
| Hybridization protocol |
The hybridization of DNA samples for ChIP-ChIP analysis was performed by NimbleGen Systems, Inc. Briefly, 13ug of the Cy5-labeled ChIP sample and 13ug of the Cy3-labeled total sample were mixed, dried down, and resuspended in 40 ul of NimbleGen Hybridization Buffer (NimbleGen Systems) plus 1.5 ug of human COT1 DNA. After denaturation, hybridization was carried out in a MAUI Hybridization System (BioMicro Systems) for 18 h at 42°C. The arrays were washed using NimbleGen Wash Buffer System (NimbleGen Systems), dried by centrifugation.
|
| Scan protocol |
The arrays were scanned at 5-um resolution using the GenePix 4000B scanner (Axon Instruments).
|
| Description |
A complete genomic analysis of KAP1 binding in human Ntera2 testicular carcinoma cells using a 38 array tiling set, identifying ~7000 KAP1 binding sites.
|
| Data processing |
log base 2 ratios; specifically, fluorescence intensity raw data were obtained from scanned images of the oligonucleotide tiling arrays using NIMBLESCAN 2.0 extraction software (NimbleGen Systems). For each spot on the array, log2-ratios of the Cy5-labeled test sample versus the Cy3-labeled reference sample were calculated. Then, the biweight mean of this log2 ratio was subtracted from each point; this procedure is approximately equivalent to mean-normalization of each channel.
|
| |
|
| Submission date |
Aug 02, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Kimberly Rose Blahnik |
| E-mail(s) |
krblahnik@ucdavis.edu
|
| Organization name |
University of California Davis
|
| Department |
Genome and Biomedical Sciences
|
| Lab |
Peggy Farnham
|
| Street address |
GBSF 1 Shields Ave
|
| City |
Davis |
| State/province |
CA |
| ZIP/Postal code |
95616 |
| Country |
USA |
| |
|
| Platform ID |
GPL5205 |
| Series (1) |
| GSE8667 |
Genome-wide Analysis of KAP1 Binding Suggests Auto-regulation of KRAB-ZNFs |
|
