|
| Status |
Public on Oct 28, 2016 |
| Title |
RWPE1 empty vector replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
RWPE1 empty vector
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: RWPE1
cell type: human prostatic epithelial cell line
genotype/variation: expressing empty vector
|
| Growth protocol |
RWPE1 cells were grown in Keratinocyte SFM media supplemented with bovine pitutiary extract, human recombinant epidermal growth factor, penicillin/streptomycin, and selection agents (puromycin/hygromycin)
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA from three biological replicates of RWPE1 cells expressing either vector, ERG-WT, ERG-P436A or ERG-WT with EWS knockdown shRNA was isolated using RNeasy kit (Qiagen) according to manufacturer’s protocol. Total RNA was DNase treated with RNase-Free DNase set (Qiagen) according to manufacturer’s protocol.
polyA containing RNA was purified using oligo(dT) beads (Invitrogen). cDNAs of polyA selected RNA was generated with SuperScript III reverse transcriptase enzyme using random hexamers (Invitrogen). Second strand synthesis from cDNAs was carried out with E. coli DNA ligase and E. coli DNA polymerase I (New England BioLabs). The double-stranded cDNAs were sheared to ~150 nucleotide fragments using a Diagenode BioRuptor and the size was confirmed by DNA gel electrophoresis.Sheared DNA was end-repaired and ligated to indexing primers provided in Truseq sample preparation kit (Illumina). DNA libraries were pooled and sequenced in Illumina NextSeq 500 according to manufacturers protocol.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Casava1.8.2 software was used for basecalling
Differential gene expression analysis was carried out with Tuxedo Suite RNA sequencing pipeline (Trapnell et al., Nature Protocol 2012). Sequence reads obtained from illumina sequencer was mapped to the human genome (hg19, UCSC) using Bowtie2. Differential gene expression was determined using Cuffdiff.
Genome_build: UCSC Hg19
Supplementary_files_format_and_content: Tab delimited text file of differential gene expression analysis. Output from CuffDiff
|
| |
|
| Submission date |
May 16, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Vivekananda Kedage |
| E-mail(s) |
vkedage@indiana.edu
|
| Organization name |
Indiana University
|
| Street address |
1001 E. third Street
|
| City |
Bloomington |
| State/province |
IN |
| ZIP/Postal code |
47405 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE81493 |
An interaction with Ewing's sarcoma breakpoint protein EWS defines the specific oncogenic mechanism of ETS factors rearranged in prostate cancer |
|
| Relations |
| BioSample |
SAMN05003736 |
| SRA |
SRX1767584 |
