|
| Status |
Public on Dec 16, 2011 |
| Title |
CD34+ sample 111 (anti-acetyl Histone H3) |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
pooled AML blasts
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: pooled AML blasts
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted and sonicated to an average DNA length of 500 bp.
|
| Label |
Cy5
|
| Label protocol |
DNA was amplified in 2 steps, including a T7 sequenase extension using random primer with a fixed sequence linker and a second step of amplification using the fixed sequence primer and Taq polymerase. The products were purified and labeled with amino-allyl-conjugated dUTP using the BioPrime labeling kit (Invitrogen, Carlsbad, CA) and random primer. Products were purified and crosslinked with monofunctional NHS-ester Cy3 or Cy5 dye.
|
| |
|
| Channel 2 |
| Source name |
CD34+ progenitor cells
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: CD34+ progenitor cells
diagnosis: CD34+
antibody: anti-acetylated Histone H3 (Upstate Biotechnology)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
2.0E+07 cells were fixed with formaldehyde, neutralized with glycine, and rinsed with cold phosphatate-buffered saline. After lysis of the cells, samples were sonicated to an average DNA length of 500 bp. Immunoprecipitation of 0.5 mg precleared chromatin was carried out by addition of 3 ug of the following antibodies: anti-acetylated Histone H3, anti-HDAC1 and anti-H3K9me3 (all antibodies obtained from Upstate Biotechnology). Only the anti-acetylated Histone H3 data is presented here. At least 2 independent ChIPs were performed for each cell line and antibody.
|
| Label |
Cy3
|
| Label protocol |
DNA was amplified in 2 steps, including a T7 sequenase extension using random primer with a fixed sequence linker and a second step of amplification using the fixed sequence primer and Taq polymerase. The products were purified and labeled with amino-allyl-conjugated dUTP using the BioPrime labeling kit (Invitrogen, Carlsbad, CA) and random primer. Products were purified and crosslinked with monofunctional NHS-ester Cy3 or Cy5 dye.
|
| |
|
| |
| Hybridization protocol |
Hybridizations were performed for 18h.
|
| Scan protocol |
Slides were scanned using an Axon 4000B scanner. Images were quantified using the SpotReader software.
|
| Data processing |
Background intensities given by the SpotReader software were subtracted from foreground intensities. The vsn method (W. Huber et al.; Bioinformatics 2002;18 Suppl 1:S96-104) was then applied for single color normalization of the green (ChIP) channel across all arrays. A model with 48 strata per array according to the print-tips was used. Probes that were flagged by the Spotreader software in more than 25% of the samples as well as empty/blank probes were excluded from the estimation of the model’s parameters. The quantile that is used for the least trimmed sum of squares regression was set to 0.75. After parameter estimation, vsn transformation was applied to all probes. All computations were done within the R-package vsn (ver. 3.18.0).
|
| |
|
| Submission date |
Mar 09, 2011 |
| Last update date |
Dec 16, 2011 |
| Contact name |
Hans-Ulrich Klein |
| E-mail(s) |
h.klein@uni-muenster.de
|
| Organization name |
Columbia University Medical Center
|
| Department |
Neurology
|
| Lab |
Center for Translational and Computational Neuroimmunology
|
| Street address |
622 W 168th St
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
| |
|
| Platform ID |
GPL10087 |
| Series (1) |
| GSE27863 |
Genome wide analysis of Histone H3 acetylation patterns in AML identifies PRDX2 as an epigenetically silenced tumor suppressor gene |
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