|
| Status |
Public on Oct 22, 2017 |
| Title |
EWS/FLI knockdown + Mut9 rescue |
| Sample type |
SRA |
| |
|
| Source name |
EWS/FLI knockdown + Mut9 rescue
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: patient-derived A673 Ewing sarcoma cells
passages: 15-18
treatment: EWS/FLI knockdown + Mut9 rescue
chip antibody: anti-FLI (sc-356X Santa Cruz Biotechnology, Inc)
|
| Treatment protocol |
A673 cells were treated with EF-2-RNAi shRNA to knock-down EWS/FLI, followed by rescue with EWS/FLI mutant cDNA (delta22 and Mut9 respectively) cloned into MSCV retroviral vectors
|
| Growth protocol |
A673 Ewing sarcoma cells with different treatment were grown to 95% confluence in 15cm culture dishes in DMEM medium supplemented with FBS and Na-Pyruvate and appropriate selection antibiotics for treatment samples
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei-DNA complexes were isolated with antibody.
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Qiagen). 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 (32 to 52 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 3’ 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 following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Basecalls performed using CASAVA
ChIP-seq reads were aligned to the hg19 genome assembly using BWA-mem
PCR duplicates were marked and removed
bigwig files were generated using deepTools and normalized to 1x genomic coverage
Genome_build: hg19
Supplementary_files_format_and_content: bigwig
|
| |
|
| Submission date |
Feb 03, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Kirsten Johnson |
| E-mail(s) |
kirsten.johnson@nationwidechildrens.org
|
| Phone |
6143552989
|
| Organization name |
Nationwide Childrens Hospital
|
| Department |
Childhood Cancer and Blood Disorders
|
| Lab |
Stephen Lessnick
|
| Street address |
The Research Institute, WA5110, 700 Childrens Drive
|
| City |
Columbus |
| State/province |
Ohio |
| ZIP/Postal code |
43205 |
| Country |
USA |
| |
|
| Platform ID |
GPL20301 |
| Series (2) |
| GSE94480 |
A novel role for the EWS portion of EWS/FLI in binding GGAA-microsatellites required for oncogenic transformation in Ewing sarcoma [ChIP-Seq] |
| GSE94503 |
A novel role for the EWS portion of EWS/FLI in binding GGAA-microsatellites required for oncogenic transformation in Ewing sarcoma |
|
| Relations |
| BioSample |
SAMN06294285 |
| SRA |
SRX2537099 |
