|
| Status |
Public on Sep 14, 2011 |
| Title |
GR_ChIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
GR_ChIP-seq
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LNCaP-1F5
cell type: Prostate epithelial cells derived from metastatic prostate carcinoma
genotype/variation: genetically engineered to express rat glucocorticoid receptor (Cleutjens et al., 1997).
passages: 15-35
chip antibody: GR
antibody vendor, cat. number: non-commercial (Widen et al, 2000)
|
| Treatment protocol |
FoxA1 depletion was done using siRNA smartpool specific to FoxA1 mRNA. Both parental and FoxA1 depleted cells were treated with 100 nM DHT (or 100 nM DEX) for 2 hours followed by Chromatin Immunoprecipitation or DNaseI-Hypersensitivity mapping.
|
| Growth protocol |
LNCaP-1F5 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 10 mM HEPES supplemented with antibiotics (penicillin and streptomycin).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and DNA-protein complexes were isolated with respective antibodies. Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3' to 5' exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3â end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols. The DNaseI-hypersensitivity assay and deep sequencing was performed as described by Song and Crawford (2010).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
Chromatin IP against GR in parental cells
|
| Data processing |
Alignment: Sequence reads were obtained and mapped to the human (Feb, 2009) genomes using the Illumina Genome Analyzer Pipeline (ELAND) or BOWTIE. All reads mapping with two or fewer mismatches were retained and read starts were summed in sliding windows of 400 bp to create summary windows.
Peaks: Peak detection was performed with the Model-based Analysis of ChIP-Seq (MACS) algorithm (http://liulab.dfci.harvard.edu/MACS/) and peaks were assigned to nearest Refseq gene using a transcription factor associtaion strength (TFAS) approach (Ouyang et al., 2009). Overlapping peaks present in both biological replicates are shown in the peak file [The TTFAS_peak files are provided as Series supplementary file]. The DHS- seq data was analyzed using F-seq algorithm (http://www.genome.duke.edu/labs/furey/software/fseq/).
|
| |
|
| Submission date |
Jul 12, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Olli A. Jänne |
| E-mail(s) |
olli.janne@helsinki.fi
|
| Phone |
+358919125040
|
| Organization name |
University of Helsinki
|
| Department |
Inst. of Biomedicine/Physiology
|
| Lab |
Androgen Receptor Laboratory
|
| Street address |
Haartmaninkatu-8
|
| City |
Helsinki |
| ZIP/Postal code |
FI-00014 |
| Country |
Finland |
| |
|
| Platform ID |
GPL9052 |
| Series (2) |
| GSE30623 |
Dual Role of FoxA1 in Androgen Receptor Binding to Chromatin, Androgen Signaling and Prostate Cancer [ChIP_seq, DHS_seq] |
| GSE30624 |
Dual Role of FoxA1 in Androgen Receptor Binding to Chromatin, Androgen Signaling and Prostate Cancer |
|
| Relations |
| SRA |
SRX083228 |
| BioSample |
SAMN00668267 |
