|
| Status |
Public on Sep 05, 2017 |
| Title |
H2AZ_ChIPseq_LNCaP_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Human cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: LNCaP clone FGC (ATCC CRL-1740). Androgen-sensitive human prostate adenocarcinoma cells
passage number: 75
chip antibody: Active Motif Cat #39113
|
| Treatment protocol |
Human LNCaP or VCaP prostate cancer cells and were maintained in RPMI Medium 1640 (Gibco), or DMEM media 11995 (Gibco), respectively, and supplemented with 10% fetal bovine serum (FBS) for at least 72 hrs to 70-80% confluence. LNCaP cell were then seeded to 2.5x106 cell per 15cm dish and starved for 3 days in RPMI Medium 1640-no-phenol red (Gibco) and 5% chracoal stripped FBS (CSFBS). Then LNCaP cells were treated with or without (vehicle, ETOH) 10nM of DHT for 2h, 4h and 16h. VCaP cell were then seeded to 2.5x106 cell per 15cm dish and starved for 3 days in DMEM media 11995 (Gibco) and 2.5% chracoal stripped FBS (CSFBS). Then VCaP cells were treated with or without (vehicle, ETOH) 10nM of DHT for 16h. Cells were then fixed with 1% formaldehyde for 10 min at room temperature.
|
| Growth protocol |
PrEC cells were growth and maintained in PrEGM media (Cambrex) at 37ºC with 5% CO2; LNCaP cells were growth and maintained in T-medium (Gibco) and 10% fetal bovine serum (FBS) at 37ºC with 5% CO2. When reached 70-80% confluence cell were fixed with 1% formaldehyde for 10 min at room temperature.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP assays were carried out according to the manufacturer's protocol (Upstate Biotechnology). Briefly, ~ 2 x 10^6 cells were fixed by adding formaldehyde at a final concentration of 1% and incubating for 10 minutes at 37°C. 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 Histone H2AZ (Active motif # 39113), acetylated Histone H2AZ (Abcam #ab18262), H3K4Me1 (Active Motif #39297), H3K27ac (Active Motif #39133), H3K9K14ac (Millipore #06-599.) and H3K36Me3 (Abcam ab9050-100). Ten ul 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 ul H2O.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Data processing |
Reads were mapped to the human genome (hg19) using Bowtie (version 1.0.1), allowing up to three mismatches. Non-uniquely alignable reads were excluded.
Peaks were called using PeakRanger (version 1.16) with peak size set to broad and with input control set to a pooled control LNCaP or VCaP input.
Bigwig files were generated using a scaling factor of 1,000,000 reads/library size, average fragment length of 300 and duplicates removed.
ChromHMM v.1.10 was used to simultaneously partition the genome of PrEC and LNCaP cell lines into states. The same approach was employed for the VCaP cell line. Redundant states were then collapsed into seven distinct states and manually annotated by comparison to the published ChromHMM model for HMEC cells.
genome_build = hg19
processed data files format and content: bed file format
|
| |
|
| Submission date |
Dec 24, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Fatima Valdes Mora |
| E-mail(s) |
[email protected]
|
| Phone |
+61 2 92958334
|
| Organization name |
Garvan Institute of Medical Research
|
| Department |
Genomics and Epigenetics
|
| Lab |
Histone Variant Group
|
| Street address |
384 Victoria Street
|
| City |
Darlinghurst |
| State/province |
NSW |
| ZIP/Postal code |
2010 |
| Country |
Australia |
| |
|
| Platform ID |
GPL9115 |
| Series (2) |
| GSE76336 |
Novel contribution of acetylated histone variant H2A.Z in activation of neo-enhancers in prostate cancer [ChIP-seq] |
| GSE76337 |
Novel contribution of acetylated histone variant H2A.Z in activation of neo-enhancers in prostate cancer |
|
| Relations |
| BioSample |
SAMN04370256 |
| SRA |
SRX1502208 |
