|
| Status |
Public on Oct 06, 2016 |
| Title |
VCaP_GROseq_EtOH_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
VCaP cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Vertebral Cancer of Prostate (VCaP)
origin: bone metastasis of prostate cancer
treatment: Ethanol
time: 2h
|
| Treatment protocol |
Steroid-depleted medium (DMEM containing 2.5% charcoal-stripped FBS) was changed to the cells 3 days before exposing the cells to vehicle (EtOH) or R1881 (10 nM) for both GRO-seq samples and ChIP-seq samples.
|
| Growth protocol |
The cells were grown in DMEM containing 10% (v/v) defined FBS, 25 U/ml penicillin and 25 mg/ml streptomycin in a 5% CO2 atmosphere at 37 C incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Nuclei were extracted from 10 million cells and nuclear run-on reactions were performed for 5 minutes at 30°C in the presence of Sarkosyl and Br-UTP. RNA was extracted with Trizol, DNase-treated, base-hydrolyzed and dephosphorylated with PNK. Br-UTP-labeled run-on RNA was immunopurified with anti-BrdU-coated agarose beads and precipitated overnight.
Poly(A)-tailing was followed by cDNA synthesis using complementary poly(T)-primers. Excess oligo was removed by Exonuclease I and cDNA fragments of were purified on a denaturing 10% polyacrylamide TBE-urea gel. The recovered cDNA was circularized, linearized, amplified with 11-14 cycles. The final product was ran on 10% TBE gel, gel purified and cleaned-up using ChIP DNA clean & Concentrator Kit. The final libraries were sequenced using an Illumina HiSeq 2000.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
nascent RNA
GRO-seq
processed data file: VCaP_gene_expression.xlsx
|
| Data processing |
The quality of raw reads was analyzed by FastQC and FASTX-toolkit was used for preprocessing raw reads (removal of artifacts, trimming reads, quality filtering and collapsing identical reads).
Reads were aligned against human genome hg19 with Bowtie software (0.12.7) with the command line: -v 1 -k 1 -m 1 -f -S --best hg19.
The ChIP-seq peak detection was done by using HOMER software (V 4.7.2; http://homer.salk.edu/homer/)). Binding sites were determined with findPeaks command (using -factor mode and IgG sample as a background). BedGraph file was done by combining two replicates and normalizing total number of reads to 10 million. GRO-seq analysis were done by using HOMER software (V 4.7.2) to determine the gene expression with analyzeRepeats command.
Genome_build: hg19
Supplementary_files_format_and_content: peaks.bed files contain information of the statistically significant peaks (found in both replicates). BedGraph file show signal intensities. Excel file of GRO-seq data show gene expression values.
|
| |
|
| Submission date |
Jul 14, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Sari Toropainen |
| E-mail(s) |
sari.toropainen@uef.fi
|
| Organization name |
University of Eastern Finland
|
| Department |
School of Medicine
|
| Lab |
Biomedicine
|
| Street address |
Yliopistonranta 1 E
|
| City |
Kuopio |
| ZIP/Postal code |
70211 |
| Country |
Finland |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE84432 |
Analysis of active enhancers and direct androgen receptor target genes in VCaP prostate cancer cells |
|
| Relations |
| BioSample |
SAMN05392191 |
| SRA |
SRX1950163 |
