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| Status |
Public on Jun 27, 2013 |
| Title |
ARmo_100nM |
| Sample type |
SRA |
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| Source name |
ARmo cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: LNCaP-ARmo
ar overexpression: 2 - 4 fold
dht treatment: 100 nM
chip antibody: AR
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| Treatment protocol |
The cells were maintained under geneticin 250 μg/ml (Invitrogen Inc., Carlsbad, CA, USA). Four million cells were plated and hormone-deprived for 4 days and treated with DHT at different concentrations for 2 h. Cells were fixed by adding formaldehyde (Merck KGaA, Darmstadt, Germany) in 1% final concentration for 10 min at room temperature and lysed in 1% sodium dodecyl sulfate, 10 mM EDTA, 50 mM Tris–HCl containing 2X protease inhibitor (Roche Inc., Mannheim, Germany). To perform tissue ChIP, 3 ml of phosphate-buffered saline containing 2X protease inhibitor (Roche Inc.) were added to 40 × 20 μm sections of freshly frozen xenograft specimens. They were first vigorously mixed three times with syringe and 14G needle, then four times with 25G needle. The cells were fixed for 10 min in room temperature by adding 1/10 volume of fixation solution (11% formaldehyde, 0.1 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM HEPES). Fixation was stopped by adding 1/20 volume of 2.5 M glycine for 5 min at room temperature. The cells were pelleted, washed twice in phosphate-buffered saline containing 2X protease inhibitor (Roche Inc.) and lysed as above. The chromatin was immunoprecipitated with 10 μg of normal rabbit immunoglobulin G (Santa Cruz Inc., Santa Cruz, CA, USA) or 10 μl of anti-AR polyclonal antibody (AR3) (provided by one of the investigators: OAJ).
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| Growth protocol |
The establishment of LNCaP cells overexpressing AR has been described previously (Waltering et al., 2009). Two PC xenografts, LuCaP69 and LuCaP73, grown in intact male mice, were provided by one of the investigators (RLV).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The chromatin was sonicated to reach a fragment size of 150-300 bp with Bioruptor UCD-200TM-EX instrument (Diagenode Inc., Liège, Belgium), diluted 1:10 in 1% Triton-X100, 2 mM EDTA, 20 mM Tris-HCl, 150 mM NaCl containing 2X protease inhibitor and precleared for 1 hour with 100 μg of yeast tRNA (Invitrogen Inc., Carlsbad, California, USA) and 20 μl of preimmune rabbit serum (Santa Cruz Inc., Santa Cruz, California, USA) followed by 1 hour incubation with 40 μl of 50% slurry of Gammabind Sepharose beads (GE Healthcare Bio-Sciences, Piscataway, New Jersey, USA) at 4°C. The beads were precipitated and 10 μg of normal rabbit IgG (Santa Cruz Inc., Santa Cruz, California, USA) or 10 μg of anti-AR (clone N20; Santa Cruz Inc., Santa Cruz, California, USA) or 10 μl of anti-AR polyclonal antibody (AR3) (provided by one of the authors: O. A. J.) (Karvonen et al., 1997, Thompson et al., 2006) were added to the supernatant for overnight incubation. The complexes were then incubated with 70 μl of Gammabind Sepharose beads (GE Healthcare Bio-Sciences, Piscataway, New Jersey, USA) 50% slurry for 4 hours and precipitated. The beads were washed sequentially for 10 min ach in TSE I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, and 150 mM NaCl), TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, and 500 mM NaCl), and buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl). Beads were then washed two times with TE buffer. The complexes were then eluted from the beads incubating for 15 minutes at 37°C twice with 1% SDS, 0.1 M NaHCO3. The eluates were pooled and treated for 1 hour at 37°C with RNAase A (Qiagen Inc., California, USA) and 1 hour with proteinase K (Finnzymes Oy, Espoo, Finland), then heated at 65°C for 6 3 hours to reverse the formaldehyde cross-linking. The DNA fragments were purified with a DNA purification kit (QIAquick PCR purification Kit, Qiagen Inc., California, USA) and eluted in 100μl of elution buffer. The libraries of ChIP DNA were prepared and sequenced with Genome analyzer II (Illumina Inc., San Diego, California, USA) according to the manufacturer’s protocol. Each sample was sequenced in one lane. The raw reads were obtained using Illumina analysis pipeline.
Standard illumina protocol
one sample per illumina lane
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Data processing |
Illumina Genome Analyzer II base calling software
The raw reads alignment was carried out with Bowtie and the reads were mapped on the human genome version 19 (hg19). Two mismatches were allowed in the seed region of the read (-n 2), with the default seed length of 28 bp. Only the best alignment was chosen.
The peak detection (i.e., binding site detection) step of the ChIP-seq analysis was performed with the tool MACS 1.3.7. using the default p-value threshold (p=0.00001), high-confidence fold-enrichment of 10 (--mfold 10) for model building and approximately half of the maximum sonication size (--bw 175) for the cell line samples and p-value threshold of 0.0001 and --mfold 5 for the xenograft samples.
Genome_build: hg19
Supplementary_files_format_and_content: "pcDNA3.1/Armo/Arhi_pool_IgG_control" and "LuCaP_pool_IgG_control" were used as controls in peak detection in for cell line and xenograft samples, respectively. The file "high_confidence_ARBSs.bed" was created by using the peaks of ARmo_1nM, ARhi_1nM and pcDNA3.1_100nM
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| Submission date |
Jun 26, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Janne Seppälä |
| E-mail(s) |
janne.seppala@tut.fi
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| Organization name |
Institute of Biomedical Technology
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| Street address |
Biokatu 8
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| City |
Tampere |
| ZIP/Postal code |
33520 |
| Country |
Finland |
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| Platform ID |
GPL9115 |
| Series (1) |
| GSE48308 |
Overexpression of androgen receptor enhances the binding of the receptor to the chromatin in prostate cancer |
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| Relations |
| BioSample |
SAMN02213913 |
| SRA |
SRX315147 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1174482_ARmo_100nM_peaks.bed.gz |
12.8 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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