|
| Status |
Public on Aug 15, 2013 |
| Title |
minusDHT_PCGEM1_hs17l4_3 |
| Sample type |
SRA |
| |
|
| Source name |
LNCaP cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: LNCaP cells
|
| Treatment protocol |
Prior to androgen treatment, we grow the cells in UltraCULTURE (Phenol red free) + 5% Charcoal Dextran Stripped (CDS) serum for 48 hours. We treat cells with DHT from a 100 uM stock in 70% ethanol to a final concentration of 100 nM.
|
| Growth protocol |
We maintain LNCaP cells in RPMI 1640 + 10% FBS medium. We typically grow LNCaP cells in 150 or 100 mm polystyrene tissue culture dishes depending on number of cells required. Cells are split soon after reach 80% confluence.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIRP was performed as described (Chu et al 2011) with minor modifications. Briefly, 60-mer antisense DNA probes targeting PRNCR1 and PCGEM1 full-length sequences were designed by http://www.singlemoleculefish.com/designer.html. A set of probes targeting LacZ RNA was also designed as the negative control. All probes were biotinylated using Label IT® Nucleic Acid Biotin Labeling Kit from Mirus Biotechnology. LNCaP cells were fixed with 1% formaldehyde for 10 min at room temperature. Crosslinking was then quenched with 125mM glycine for 5 min. The chromatin preparation, hybridization/elution, deep sequencing steps were essentially performed as described (Chu et al 2011) except that washing was conducted at 50 °C and 65°C.
ChIRP complexes were washed and the DNA was extracted and purified by QIAquick Spin columns (Qiagen). For ChIRP-seq, extracted DNA was ligated to specific adaptors followed by deep sequencing.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
ChIRP-seq
|
| Data processing |
library strategy: ChIRP-seq
The image analysis and base calling were performed by using Illumina's Genome Analysis pipeline.
The sequencing reads were aligned to hg18 using Bowtie2, and we kept only 1 aligned read/ genome position for downstream analyses.
The bedgraph files were generated by using HOMER.
The peak finding was carried by using HOMER.
The motif enrichment analysis was carried by using HOMER.
Genome_build: hg18
Supplementary_files_format_and_content: BedGraph files display of continuous-valued data in track format, and were generated by using the HOMER suite ("makeUCSCfile" command).
Supplementary_files_format_and_content: BED file after aligning the reads to hg18 by using Bowtie2, keeping only 1 mapped read / genome position
Supplementary_files_format_and_content: HOMERpeak files display the coordinates of the peaks, as these were predicted by the HOMER suite ("findPeaks" command).
|
| |
|
| Submission date |
Jun 10, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
MICHAEL G ROSENFELD |
| E-mail(s) |
mrosenfeld@ucsd.edu
|
| Organization name |
HHMI/UCSD
|
| Street address |
9500 Gilman Drive
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE47804 |
LncRNA-dependent mechanisms of androgen receptor-regulated gene activation programs [ChIRP-seq] |
| GSE47807 |
LncRNA-dependent mechanisms of androgen receptor-regulated gene activation programs |
|
| Relations |
| BioSample |
SAMN02194345 |
| SRA |
SRX298678 |
