|
| Status |
Public on Jun 02, 2009 |
| Title |
Hypoxia-HIF1-6 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
IP: HepG2 cells under hypoxic condition (0.5%O2, 4 hours)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HepG2
cell type: hepatocellular carcinoma
cell tissue: liver
antibody: HIF-1 polyclonal
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
HepG2 cells were cultured under normoxic (ambient) or hypoxic (0.5% O2, 4 h) conditions. Cells were cross-linked with 1% formaldehyde for 10 minutes at 37ºC, washed with cold PBS, and lysed with 0.5% SDS Buffer. Chromatins were then sonicated in 0.5% SDS Buffer using 3 x 10 second pulses on setting 5 with a Fisher Scientific Sonic Dismembrator (model 100) into 500bp~1kb fragments. Lysates were then diluted with dilution buffer into final SDS concentration being 0.1%. Three ChIP biological replicates were performed for both normoxic and hypoxic samples with HIF-1a pAb (Novus, NB 100-134). A small aliquot for each biological sample are reserved for an input control. Both Chiped DNA and Input samples were amplified by LM-PCR.
|
| Label |
biotin
|
| Label protocol |
Amplified ChIP and Input samples were fragmented with DNase to 50~100bp size and labeled using the Terminal transferase (TdT, Promega) to add biotinylated-ddATP to the ends of the fragments.
|
| |
|
| Channel 2 |
| Source name |
Input: HepG2 cells under hypoxic condition (0.5%O2, 4 hours)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HepG2
cell type: hepatocellular carcinoma
cell tissue: liver
antibody: none
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
HepG2 cells were cultured under normoxic (ambient) or hypoxic (0.5% O2, 4 h) conditions. Cells were cross-linked with 1% formaldehyde for 10 minutes at 37ºC, washed with cold PBS, and lysed with 0.5% SDS Buffer. Chromatins were then sonicated in 0.5% SDS Buffer using 3 x 10 second pulses on setting 5 with a Fisher Scientific Sonic Dismembrator (model 100) into 500bp~1kb fragments. Lysates were then diluted with dilution buffer into final SDS concentration being 0.1%. Three ChIP biological replicates were performed for both normoxic and hypoxic samples with HIF-1a pAb (Novus, NB 100-134). A small aliquot for each biological sample are reserved for an input control. Both Chiped DNA and Input samples were amplified by LM-PCR.
|
| Label |
biotin
|
| Label protocol |
Amplified ChIP and Input samples were fragmented with DNase to 50~100bp size and labeled using the Terminal transferase (TdT, Promega) to add biotinylated-ddATP to the ends of the fragments.
|
| |
|
| |
| Hybridization protocol |
Approximately 2ug of DNA was hybridzed per array.
|
| Scan protocol |
Both hybridization and scan were performed by Microarray Core at Dana-Farber Cancer Institute.
|
| Description |
HIF-1 ChIP Hypoxia Biological Rep 1-3, Affymetrix Human Tiling 2.0R Set, Array 6
|
| Data processing |
The model-based analysis of tiling-array (MAT) algorithm was used to identify regions enriched by ChIP-chip (‘‘hits’’) on Affymetrix whole-genome or promoter tiling arrays. MAT was used to identify probe intensity peaks comparing triplicate hypoxic ChIP vs. hypoxic input (‘‘Hypoxic’’) and normoxic ChIP vs. normoxic input (‘‘Normoxic’’). MAT was run with the same parameters for each: bandwidth = 200, maximum gap = 400, minimum probes = 10, and P value cutoff = 1e - 5. Hits appearing solely in the hypoxic samples were retained as hypoxia-unique. In cases where hypoxic and normoxic hits overlapped, hits for which the MAT score of hypoxic subtracted by that of normoxic samples were above the cutoff were also retained. The MAT library and mapping files were based on the March 2006 Human Genome Assembly (HG18). Hits flagged by MAT as mapping to repeat regions were excluded from consideration.
|
| |
|
| Submission date |
May 29, 2009 |
| Last update date |
Jun 01, 2009 |
| Contact name |
Xiaobo Xia |
| E-mail(s) |
Xiaobo_Xia@dfci.harvard.edu
|
| Organization name |
Dana-Farber Cancer Institute
|
| Department |
Pediatric Oncology
|
| Lab |
Dr. Andrew Kung
|
| Street address |
44 Binney St, Mayer 649
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL4915 |
| Series (1) |
| GSE16347 |
Genome-wide HIF-1 binding sites in HepG2 cells |
|
