|
Status |
Public on Oct 09, 2009 |
Title |
HepG2-RNApolII ChIP-chip |
Sample type |
genomic |
|
|
Channel 1 |
Source name |
HepG2 RNA polII ChIP Normoxia
|
Organism |
Homo sapiens |
Characteristics |
antibody: RNA polymerase II mAb (Abcam, ab5408) cell line: HepG2 cells stress: normoxic (ambient) conditions
|
Treatment protocol |
U87 cells were cultured for 4 hours under normoxic or hypoxic (0.5%O2) conditions.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Cells were fixed with 1% formaldehyde (37°C, 10 min) and lysed with 0.5% SDS lysis buffer. Chromatin was then sonicated to 500-1000bp fragments and immunoprecipitation carried out with HIF-1α pAb (Novus Biologicals, NB 100-134). RNA polymerase II and H3K4me3 ChIP were carried out using normoxic U87 or HepG2 cell samples with RNA polymerase II mAb (Abcam, ab5408) and H3K4me3 pAb (Abcam, ab8580). 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 |
HepG2 RNA polII input Normoxia
|
Organism |
Homo sapiens |
Characteristics |
cell line: HepG2 cells stress: normoxic (ambient) conditions antibody: none
|
Treatment protocol |
U87 cells were cultured for 4 hours under normoxic or hypoxic (0.5%O2) conditions.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Cells were fixed with 1% formaldehyde (37°C, 10 min) and lysed with 0.5% SDS lysis buffer. Chromatin was then sonicated to 500-1000bp fragments and immunoprecipitation carried out with HIF-1α pAb (Novus Biologicals, NB 100-134). RNA polymerase II and H3K4me3 ChIP were carried out using normoxic U87 or HepG2 cell samples with RNA polymerase II mAb (Abcam, ab5408) and H3K4me3 pAb (Abcam, ab8580). 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 3ug of DNA was hybridzed per array.
|
Scan protocol |
Both hybridization and scan were performed by Microarray Core at Dana-Farber Cancer Institute.
|
Description |
HepG2 RNA polII ChIP Normoxia Biological Rep 1-3 v.s. Inputs, Affymetrix Human Promoter 1.0R Array test CEL file: HepG2_PolII_C1.CEL test CEL file: HepG2_PolII_C2.CEL test CEL file: HepG2_PolII_C3.CEL control CEL file: HepG2_NI1.CEL control CEL file: HepG2_NI2.CEL control CEL file: HepG2_NI3.CEL
|
Data processing |
The Model-based Analysis of Tiling-array (MAT) algorithm was used to identify regions enriched by ChIP-chip (ChIP hits). For the U87 HIF-1 ChIP, the triplicate hypoxic U87 HIF-1 ChIP samples were compared directly to triplicate normoxic samples; MAT was run with parameters of bandwidth = 200, maximum gap = 400, minimum probes = 10, and p-value cutoff = 1x10-5. For H3K4me3, H3K27me3, and RNA Pol II ChIP, normoxic ChIP samples were compared to matched input samples; the MAT parameters were increased to account for broader peaks: bandwidth = 500, maximum gap = 400, minimum probes = 20, and p-value cutoff = 1x10-5. 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 in all cases.
|
|
|
Submission date |
Oct 09, 2009 |
Last update date |
Oct 09, 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 |
GPL5082 |
Series (2) |
GSE18499 |
Genome-wide HIF-1, RNA polII, and H3K4me3 binding sites in U87 and HepG2 cells |
GSE18505 |
HepG2 hepatoma and U87 glioma cells: transcriptomic and genomic analyses |
|
Supplementary file |
Size |
Download |
File type/resource |
GSM461102_HepG2_NI1.CEL.gz |
19.5 Mb |
(ftp)(http) |
CEL |
GSM461102_HepG2_NI2.CEL.gz |
18.9 Mb |
(ftp)(http) |
CEL |
GSM461102_HepG2_NI3.CEL.gz |
19.2 Mb |
(ftp)(http) |
CEL |
GSM461102_HepG2_PolII_C1.CEL.gz |
20.4 Mb |
(ftp)(http) |
CEL |
GSM461102_HepG2_PolII_C2.CEL.gz |
20.5 Mb |
(ftp)(http) |
CEL |
GSM461102_HepG2_PolII_C3.CEL.gz |
19.3 Mb |
(ftp)(http) |
CEL |
GSM461102_HepG2_polII.Hs_PromPR_v01-3_NCBIv36.NR.bpmap_matscore.bar.gz |
20.6 Mb |
(ftp)(http) |
BAR |
GSM461102_HepG2_polII.bed.gz |
119.3 Kb |
(ftp)(http) |
BED |
Processed data included within Sample table |
Processed data provided as supplementary file |