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| Status |
Public on Jan 01, 2015 |
| Title |
LNCaP control [ChIP-exo replicate 2 ] |
| Sample type |
SRA |
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| Source name |
LNCaP cells
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| Organism |
Homo sapiens |
| Characteristics |
agent: vehicle, control
cell line: LNCaP
tissue: Prostate adenocarcinoma
type: Androgen-sensitive
chip antibody: anti-AR (N20) from Santa Cruz Biotechnology (Santa Cruz, CA)
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| Treatment protocol |
For hormone responsive experiments, LNCaP cells were maintained in phenol red-free RPMI medium with 5% charcoal-stripped FBS for 3 days, and then treated with 10 nM DHT.
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| Growth protocol |
The androgen-dependent prostate cancer cell line LNCaP was obtained from the American Type Culture Collection, and cultured in the RPMI complete medium.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For LNCaP AR ChIP-exo, after treatment with 10 nm DHT or vehicle for 4 h, LNCaP cells were crosslinked with 1% formaldehyde for 10 min. Sonicated chromatin was enriched by immunoprecipatation with an anti-AR antibody. For tissue AR ChIP-exo, the frozen samples were trimmed and chopped into small pieces on ice, and then fixed immediately in 1% formaldehyde for 20 min at room temperature. After additional incubation with glycine for 10 min, the pellets were washed and homogenized in 1x PBS containing proteinase inhibitor. With sufficient lysis, the samples were then processed according to ChIP-exo procedures. For RNA-seq, total RNA was isolated using an RNeasy kit (QIAGEN). Next, total RNA was mixed with the Ribo-Zero rRNA removal reagents (Epicentre Biotechnologies, Madison, WI) to remove all sizes of rRNA. After two rounds of removal processes, the rRNA-depleted RNA was recovered for library generation.
For AR ChIP-exo, T4 DNA polymerase, T4 PNK, and Klenow DNA Polymerase were used together for end polishing. The ligation step was performed with less reducing agent. Protein A Dynal magnetic beads were washed using modified RIPA buffer (50mM Tris-HCL pH 7.8, 1mM EDTA, 0.25% Na Deoxycholate, 1% NP-40, 0.5M LiCl). The library was amplified with only 10 or 12 PCR cycles, and prepared without gel-based size selection. For RNA-seq, the ScriptSeq library preparation was performed with ScripSeq RNA-Seq Library Preparation kit following the manufacturer’s instructions. The di-tagged cDNA molecules were amplified by 8 cycles of PCR. Non-size selection libraries were then sequenced for pair-end using Illumina HiSeq 2000 at the OSUCCC sequencing core.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
ChIP-exo replicate 2 read1 and 2
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| Data processing |
ChIP-exo Alignment: Sequence reads were obtained and mapped to the human (hg19) genomes using the Bowtie. All reads mapping with two or fewer mismatches were retained. Only reads with unique mapping location is used for downstream analysis.
RNA-seq process: The RNA-seq data was aligned by RUM. Paired-end reads were mapped to the reference genome build (hg19). The RPKM (Reads Per Kilobase of exon model per Million mapped reads) count was calculated with RseQC. The reads count in exon regions was used to estimate the expression level of the genes. edgeR (empirical analysis of digital gene expression in R) was applied to conduct the differential expression analysis based on the count information. The differentially expressed genes were detected by a cut-off P-value < 0.01 between DHT treated and control data.
Genome_build: hg19
Supplementary_files_format_and_content: Bowtie alignment files and four column mapped reads information files for ChIP-exo data; edgeR differential expressed gene files for RNA-seq data.
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| Submission date |
Jan 27, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Xun Lan |
| E-mail(s) |
xlan@stanford.edu
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| Phone |
7738345917
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| Organization name |
Stanford University
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| Department |
Genetics
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| Lab |
Pritchard's Lab
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| Street address |
318 Campus Dr. Room S240
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE43785 |
Redefinition of Human Androgen Responsive Elements [ChIP-Seq, RNA-Seq] |
| GSE43791 |
Redefinition of Human Androgen Responsive Elements |
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| Relations |
| SRA |
SRX220076 |
| BioSample |
SAMN01906558 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1071277_120224_s2_NoIndex_L002_R1.fastq.tagAlign.gz |
6.1 Gb |
(ftp)(http) |
TAGALIGN |
| GSM1071277_120224_s2_NoIndex_L002_R2.fastq.tagAlign.gz |
5.9 Gb |
(ftp)(http) |
TAGALIGN |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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