|
| Status |
Public on Jul 08, 2010 |
| Title |
DHT 6 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
AR ChIP DNA from primary human muscle cells treated with 30nM DHT for 8 hours
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Primary Human Muscle Cells
antibody: AR antibody
antibody manufacturer: Santa Cruz
antibody catalog #: sc-13062
lot #: L0704
treatment: DHT
|
| Treatment protocol |
Primary human skeletal myoblasts were treated with 30nM dihydrotestosterone (DHT) or an equal volume of DMSO for 8 hours. Treated cells were then fixed with 1% formaldehyde for 15 minutes and quenched with 0.125 M glycine.
|
| Growth protocol |
Primary human skeletal muscle myoblasts (Lonza) were grown and maintained in SkGM-2 media (Lonza) following the manufacturer’s recommendations. For TaqMan experiments, proliferating cells were grown in modified SkGM-2 media lacking dexamethasone and containing 5% charcoal-stripped fetal bovine serum. Cells were differentiated in DMEM-F12 supplemented with 2% horse serum (Lonza) following the manufacturer’s recommendations.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was isolated by adding lysis buffer, followed by disruption with a Dounce homogenizer (cells). Lysates were sonicated and the DNA sheared to an average length of 300-500bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (20-30 mg) was then pre-cleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using an antibody against AR (sc-13062, Santa Cruz Biotechnology, Inc.) or rabbit IgG (I-5006, Sigma) as a control. After incubation at 4°C overnight, protein A agarose beads were used to isolate the immune complexes. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation.
|
| Label |
biotin
|
| Label protocol |
Control and AR ChIPs and subsequent genome tiling array hybridization were performed by Genpathway, Inc. (San Diego, CA).
|
| |
|
| Channel 2 |
| Source name |
Primary Human Muscle Cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Primary Human Muscle Cells
sample type: input DNA
|
| Treatment protocol |
Primary human skeletal myoblasts were treated with 30nM dihydrotestosterone (DHT) or an equal volume of DMSO for 8 hours. Treated cells were then fixed with 1% formaldehyde for 15 minutes and quenched with 0.125 M glycine.
|
| Growth protocol |
Primary human skeletal muscle myoblasts (Lonza) were grown and maintained in SkGM-2 media (Lonza) following the manufacturer’s recommendations. For TaqMan experiments, proliferating cells were grown in modified SkGM-2 media lacking dexamethasone and containing 5% charcoal-stripped fetal bovine serum. Cells were differentiated in DMEM-F12 supplemented with 2% horse serum (Lonza) following the manufacturer’s recommendations.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was isolated by adding lysis buffer, followed by disruption with a Dounce homogenizer (cells). Lysates were sonicated and the DNA sheared to an average length of 300-500bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (20-30 mg) was then pre-cleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using an antibody against AR (sc-13062, Santa Cruz Biotechnology, Inc.) or rabbit IgG (I-5006, Sigma) as a control. After incubation at 4°C overnight, protein A agarose beads were used to isolate the immune complexes. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation.
|
| Label |
biotin
|
| Label protocol |
Control and AR ChIPs and subsequent genome tiling array hybridization were performed by Genpathway, Inc. (San Diego, CA).
|
| |
|
| |
| Hybridization protocol |
Control and AR ChIPs and subsequent genome tiling array hybridization were performed by Genpathway, Inc. (San Diego, CA).
|
| Scan protocol |
Arrays were scanned, and analyzed using Affymetrix Tiling Analysis Software (TAS)
|
| Description |
chip F
|
| Data processing |
Arrays were scanned, and analyzed using Affymetrix Tiling Analysis Software (TAS), bed files map to hg18
|
| |
|
| Submission date |
Jun 01, 2010 |
| Last update date |
Jul 08, 2010 |
| Contact name |
Yuchen Bai |
| Organization name |
Pfizer
|
| Department |
CIBB
|
| Lab |
Biolomics
|
| Street address |
500 Arcola Road, S2316
|
| City |
Collegeville |
| State/province |
PA |
| ZIP/Postal code |
19426 |
| Country |
USA |
| |
|
| Platform ID |
GPL4915 |
| Series (1) |
| GSE22076 |
The Androgen Receptor Modulates Expression of Genes with Critical Roles in Muscle Development and Function |
|
