|
| Status |
Public on May 02, 2014 |
| Title |
Mock ChIP With TSA Treatment |
| Sample type |
SRA |
| |
|
| Source name |
Human primary endothelial colony-forming cells
|
| Organism |
Homo sapiens |
| Characteristics |
source: ECFCs derived from human umbilical cord blood
antibody: isotype IgG control
treatment: 100 ng/ml final TSA
|
| Treatment protocol |
ECFCs in complete EGM-2 medium at 70% confluency were treated with 100 ng/ml final TSA (cat# 19-138; Upstate Millipore) or a vehicle control as indicated. Cells were washed with PBS prior to harvesting and crosslinked with formaldehyde for 30 min. at RT as previously described (Palii, 2011).
|
| Growth protocol |
Human umbilical cord blood from normal full-term deliveries were obtained after informed consent. Light-density mononuclear cell (MNC) fractions were isolated by Ficoll density and resuspended in complete EGM-2 medium (EBM-2 Endothelial Cell basal Medium-2, Lonza, Cat# CC-3156, supplemented with EGM-2 SingleQuot Kit Supplements & Growth Factors, Lonza, Cat# CC-4176) and 10% fetal bovine serum and plated on to CellBIND 6 well plates at 1x107 cells/well. Individual ECFC colonies were isolated with 0.05% trypsin and expanded in EGM-2 medium. ECFCs at passage 3 were used.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Binding of TAL1 transcription factor was measured using a ChIP protocol as previously described (Palii, 2011). Chromatin from 0.5x10^8 cells was fragmented to a size range of 100-300 bases with a Bioruptor (Diagenode) at 4°C. Solubilized chromatin was immunoprecipitated with an antibody against TAL1 (C-21, Santa-Cruz) or an isotype IgG control. Antibody-chromatin complexes were pulled-down using Dynabeads-Protein G, washed and eluted. After cross-link reversal and proteinase K treatment, immunoprecipitated DNA was extracted with phenol-chloroform.
ChIPed DNA was amplified using the Illumina protocol with modifications described in (Palii, 2011).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Mock ChIP with TSA Treatment
|
| Data processing |
Basecalling was performed with Illumina RTA 1.12.4.2
ChIP-Seq reads were aligned to hg19 genome assembly using BWA 0.6.2 using default parameters.
Reads not mapping to the genome, or mapping more than once to the reference genome were removed for followup analysis.
Duplicate unique reads were removed with samtools version 0.1.18 rmdup tool.
Comparative binding analysis was done by calling peaks with MACS using low stringency in each ChIP and then comparing the read tag density for each corresponding peak (as described in Palii et al. submitted). Peaks were only used to do the comparative analysis between Control and TSA treatment ChIP-Seq.
Genome_build: hg19
Supplementary_files_format_and_content: bigWIG files represent the read coverage of the ChIP-Seq across the genome. The files were generated using the BEDTools suite (version 2.16.2) and UCSC genome browser standalone tools for Unix from the aligned reads output of BWA 0.6.2 using default values.
|
| |
|
| Submission date |
Dec 17, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Marjorie Brand |
| E-mail(s) |
karthi.sivaraman@gmail.com, mbrand@ohri.ca
|
| Organization name |
Ottawa Hospital Research Institute
|
| Street address |
501 Smyth Road
|
| City |
Ottawa |
| State/province |
Ontario |
| ZIP/Postal code |
K1H 8L6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE44546 |
TAL1 in human Endothelial Colony-Forming Cells |
| GSE53423 |
Comparative analysis of TAL1 binding in TSA-treated versus non-treated Endothelial Colony Forming Cells (ECFCs) |
|
| Relations |
| BioSample |
SAMN02463321 |
| SRA |
SRX393517 |
