|
| Status |
Public on Jan 04, 2010 |
| Title |
LNCaP H3K27me3 ChIP-chip |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
LNCaP H3K27me3 ChIP DNA
|
| Organism |
Homo sapiens |
| Characteristics |
tissue type: : Prostate
cell type: Cancer cell line
growth medium: T-medium w/ 10% FCS
antibody: H3K27me3 Ab
|
| Growth protocol |
Cells were grown at 37ºC with 5% CO2
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP assays were carried out according to the manufacturer’s protocol_ch1(Upstate Biotechnology). Briefly, ~ 1 x 106 cells, in a 10 cm dish, were fixed by adding formaldehyde at a final concentration of 1% and incubating for 10 minutes at 37C. The cells were washed twice with ice cold PBS containing protease inhibitors (1mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml aprotinin and 1µg/ml pepstatin A), harvested and treated with SDS lysis buffer for 10 min on ice. The resulting lysates were sonicated to shear the DNA to fragment lengths of 200 to 500 basepairs. The complexes were immunoprecipitated with antibodies specific for acetylated-histone H3 lysine 9 (H3K9ac) (Millipore #06-599), trimethylation –histone H3 lysine 4 (H3K4me3) (Abcam #ab8580), dimethyl-histone H3 lysine 9 (H3K9me2) (Abcam #ab1220) and tri-methyl-histone H3 lysine 27 (H3K27me3) (Millipore #07-449). Ten µl of antibody was used for each immunoprecipitation. No antibody controls were also included for each ChIP assay and no precipitation was observed by quantitative Real-Time PCR (qPCR) analysis. Input samples were processed in parallel. The antibody/protein complexes were collected by either salmon sperm DNA/protein A agarose slurry or Protein A/G PLUS agarose beads (Santa Cruz sc-2003) and washed several times. The immune complexes were eluted with 1% SDS and 0.1 M NaHCO3 and samples were treated with proteinase K for 1 hour and DNA was purified by phenol/chloroform extraction, ethanol precipitation and resuspended in 30 µl H2O.
Immunoprecipitated and Input DNA was amplified with 14 cycles of whole genome amplification (WGA2 kit, Sigma) according to manufacturers instructions.
|
| Label |
biotin
|
| Label protocol |
Samples (7.5μg of DNA) were enzymatically fragmented and labeled using the WT Labeling Kit (Affymetrix) to add biotinylated compound to the ends of the fragments
|
| |
|
| Channel 2 |
| Source name |
LNCaP Input Chromatin
|
| Organism |
Homo sapiens |
| Characteristics |
tissue type: Prostate
cell type: Cancer cell line
growth medium: T-medium w/ 10% FCS
antibody: none
|
| Growth protocol |
Cells were grown at 37ºC with 5% CO2
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP assays were carried out according to the manufacturer’s protocol_ch2(Upstate Biotechnology). Briefly, ~ 1 x 106 cells, in a 10 cm dish, were fixed by adding formaldehyde at a final concentration of 1% and incubating for 10 minutes at 37C. The cells were washed twice with ice cold PBS containing protease inhibitors (1mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml aprotinin and 1µg/ml pepstatin A), harvested and treated with SDS lysis buffer for 10 min on ice. The resulting lysates were sonicated to shear the DNA to fragment lengths of 200 to 500 basepairs. The complexes were immunoprecipitated with antibodies specific for acetylated-histone H3 lysine 9 (H3K9ac) (Millipore #06-599), trimethylation –histone H3 lysine 4 (H3K4me3) (Abcam #ab8580), dimethyl-histone H3 lysine 9 (H3K9me2) (Abcam #ab1220) and tri-methyl-histone H3 lysine 27 (H3K27me3) (Millipore #07-449). Ten µl of antibody was used for each immunoprecipitation. No antibody controls were also included for each ChIP assay and no precipitation was observed by quantitative Real-Time PCR (qPCR) analysis. Input samples were processed in parallel. The antibody/protein complexes were collected by either salmon sperm DNA/protein A agarose slurry or Protein A/G PLUS agarose beads (Santa Cruz sc-2003) and washed several times. The immune complexes were eluted with 1% SDS and 0.1 M NaHCO3 and samples were treated with proteinase K for 1 hour and DNA was purified by phenol/chloroform extraction, ethanol precipitation and resuspended in 30 µl H2O.
Immunoprecipitated and Input DNA was amplified with 14 cycles of whole genome amplification (WGA2 kit, Sigma) according to manufacturers instructions.
|
| Label |
biotin
|
| Label protocol |
Samples (7.5μg of DNA) were enzymatically fragmented and labeled using the WT Labeling Kit (Affymetrix) to add biotinylated compound to the ends of the fragments
|
| |
|
| |
| Hybridization protocol |
As per standard Affymetrix protocol for the Human Promoter 1.0R tiling arrays
|
| Scan protocol |
As per standard Affymetrix protocol for the Human Promoter 1.0R tiling arrays
|
| Description |
LNCaP H3K27me3 ChIP-chip on promoter array
|
| Data processing |
All analysis was performed with UCSC hg18 version of the human genome. The model-based analysis of tiling array (MAT) algorithm (Johnson WE et al PNAS 2006) was used with a bandwidth of 1kb to generate the bar files.
|
| |
|
| Submission date |
Dec 22, 2009 |
| Last update date |
Jan 04, 2010 |
| Contact name |
Aaron Statham |
| E-mail(s) |
a.statham@garvan.org.au
|
| Organization name |
Garvan Institute of Medical Research
|
| Department |
Cancer Department
|
| Lab |
Epigenetics Research Laboratory
|
| Street address |
384 Victoria St
|
| City |
Darlinghurst |
| State/province |
NSW |
| ZIP/Postal code |
2010 |
| Country |
Australia |
| |
|
| Platform ID |
GPL5082 |
| Series (2) |
| GSE19600 |
MeDIP and ChIP-on-chip data from normal Prostate epithelial cells (PrEC) and the LNCaP cancer cell line |
| GSE19726 |
Expression, MeDIP and ChIP-on-chip data from normal Prostate epithelial cells (PrEC) and the LNCaP cancer cell line |
|
