|
| Status |
Public on Oct 30, 2014 |
| Title |
ATAC-Seq MSC pLIV empty |
| Sample type |
SRA |
| |
|
| Source name |
MSC
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Mesenchymal Stem Cells
treatment: empty vector
|
| Treatment protocol |
EWS-FLI1 was expressed using a lentiviral vector (pLIV). Lentiviruses were produced using standard protocols. Briefly, lentiviral shRNA or expression plasmids were co-transfected with GAG/POL and VSV plasmids into 293T packaging cells using FugeneHD (Roche) to produce the virus. Viral supernatant was collected 72 hours after transfection and concentrated by ultracentrifugation using an SW41Ti rotor (Beckman Coulter) at 28,000 rpm for 120 min.
|
| Growth protocol |
The human Ewing sarcoma cell lines A673 and SKMNC were grown in RPMI 1640 containing 10% FCS at 37°C with 5% CO2. Cells were maintained between a density of 5 x 10^5 cells/ml and 2 x 10^6 cells/ml and split every 3 – 4 days. Primary pediatric mesenchymal stem cells were cultured in IMDM containing 10% FCS and 10ng/ml PDGF-BB (Peprotec).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
1x10^5 cells for each condition were resuspended in lysis buffer for 10 minutes, washed, and incubated in Transposase reaction mix for 30 minutes at 37 °C (Nextera DNA sample Prep Kit, Illumina).
DNA was purified and adaptor sequences were added to the fragmented DNA by PCR to construct Illumina sequencing libraries.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
MSC.empty.ATACseq
|
| Data processing |
Library strategy: ATAC-seq
Paired end reads were aligned to the reference genome using BWA 0.6.2
Read start sites were adjusted to represent the center of the transposon binding event (+4 bp in the plus strand and -5 bp in the minus strand). Cut site density was calculated over 150bp-wide sliding windows at 20bp steps using Bedtools. Each experiment was normalized to 10M reads.
Genome_build: hg19
Supplementary_files_format_and_content: Bigwig files of ATAC-Seq cut site density
|
| |
|
| Submission date |
Oct 01, 2014 |
| Last update date |
Oct 30, 2014 |
| Contact name |
Martin Aryee |
| E-mail(s) |
aryee.martin@mgh.harvard.edu
|
| Organization name |
Massachusetts General Hospital
|
| Department |
Pathology
|
| Street address |
149 13th Street
|
| City |
Charlestown |
| State/province |
MA |
| ZIP/Postal code |
02129 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE61951 |
Genome-wide chromatin analysis of Ewing sarcoma (ATAC-seq) |
| GSE61953 |
Genome-wide chromatin analysis of Ewing sarcoma |
|
| Relations |
| BioSample |
SAMN03085613 |
