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| Status |
Public on Feb 25, 2016 |
| Title |
HB_DHS |
| Sample type |
SRA |
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| Source name |
Hemangioblast
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| Organism |
Mus musculus |
| Characteristics |
strain: 129
cell type: ES derived Hemangioblasts (Bry.GFP+, FLK1+)
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| Growth protocol |
A mouse ES cell line carrying a brachyury GFP+ reporter gene (Fehling et al, 2003, Development 130, 4217-4227) was cultured on MEFs then differentiated as described previously (Sroczynska et al, 2009, Methods Mol Biol 538, 317-334.). Both GFP and cell surface markers were used to isolate each cell population. After differentiation of ESC into embryoid bodies, mesodermal cells were isolated by FACs sorting GFP/Brachyury (Bry+, Flk1- negative cells. A proportion of these cells were allowed to differentiate towards hemangioblasts (HBs, Bry+/Flk1+), hemogenic endothelium (HEs, Tie2+/cKit+/CD41-) and haematopoietic progenitors (HPs, CD41+). Macrophages were isolated by terminal differentiation of CD41+ cells to those expressing the macrophage marker CD11b.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
At least 1x 10^6 up tp 3x 10^6 freshly sorted and thoroughly counted cells were resuspended at 3x 10^6 cells per 100 µl DNase I buffer (60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 10 mM Tris pH 7.4, 0.3 M sucrose), mixed with an equal volume of DNase I NP-40 buffer (60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 10 mM Tris pH 7.4, 0.3 M sucrose, 0.4% NP-40, 2 mM CaCl2) with suitable 2-fold concentration of DNase I enzyme (final concentration ranging between 15 U and 260 U) and incubated at 22°C for 3 min. The reaction was stopped by addition of 2x volume of lysis buffer (300 mM NaAcetate, 10 mM EDTA pH 7.4, 1% SDS, 1 mg/ml proteinase K) and samples were incubated over night at 45°C. After phenol-phenol/chloroform-chloroform extraction DNA was precipitated with 2x volume of 100% ethanol, 10 µg of digested DNA were run on a 1.2% agarose gel in TAE buffer, 50 - 300 bp fragments were size-selected and purified using Qiagen MinElute gel extraction kit. Before library preparation the DNase I digested and size-selected material was validated by realtime PCR analysis.
For library preparation ChIP material was end-repaired, respective adaptors were ligated and fragments were PCR-amplified with 9 to 18 cycles. Size-selection and adaptor-dimer removal was done via gel purification or Ampure bead purification (for SOLiD libraries only). Libraries were validated using realtime PCR for known target/accessible sites, quality was assessed by Agilent Technologies 2100 Bioanalyser and in case of multiplexing the quantity of libraries was measured by the Kapa Library Quantification Kit.
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| Library strategy |
DNase-Hypersensitivity |
| Library source |
genomic |
| Library selection |
DNAse |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
The sequence reads in fastq format were mapped to the mm10 mouse genome build using Bowtie. The resulting alignment files were used to generate density maps using HOMER.
DNaseI Hypersensitive Sites are the enriched sites obtained using DFilter (default parameters)
Genome_build: mm10
Supplementary_files_format_and_content: fastq (unmapped reads), bigWig (density profile of mapped reads) and bed (peaks)
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| Submission date |
May 20, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
MS Vijayabaskar |
| E-mail(s) |
fbsvbm@leeds.ac.uk
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| Organization name |
University of Leeds
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| Department |
School of Molecular and Cellular Biology
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| Street address |
Woodhouse Lane
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| City |
Leeds |
| ZIP/Postal code |
LS2 9JT |
| Country |
United Kingdom |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE69095 |
Comprehensive Epigenomic Analysis Reveals Dynamic Regulatory Programs Of Blood Development (Dnase-Hypersensitivity) |
| GSE69101 |
Comprehensive Epigenomic Analysis Reveals Dynamic Regulatory Programs Of Blood Development |
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| Relations |
| BioSample |
SAMN03703145 |
| SRA |
SRX1034676 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1692782_HB_DHS.bed.gz |
168.6 Kb |
(ftp)(http) |
BED |
| GSM1692782_HB_DHS.bw |
297.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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