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| Status |
Public on Feb 25, 2016 |
| Title |
HE_H3K27me3 |
| Sample type |
SRA |
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| Source name |
Hemogenic Endothelium
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| Organism |
Mus musculus |
| Characteristics |
strain: 129
cell type: ES derived Hemogenic endothelium (Bry.GFP+, Tie2+/Ckit+/CD41-)
chip antibody: H3K27me3 Abcam ab6002
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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 |
For histone modification ChIP, nuclei of approximately 2x 10^6 sorted and crosslinked cells were isolated in hypotonic buffer A (10 mM Hepes, pH 8.0, 10 mM EDTA, 0.5 mM EGTA, 0.25 % TritonX100) and washed in buffer B (10 mM Hepes, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.25 % TritonX100). Chromatin was then sonicated in immunoprecipitation buffer I (25 mM Tris 1 M, pH 8.0, 150 mM NaCl, 2 mM EDTA, pH 8.0, 1 % TritonX100 and 0.25 % SDS) using a Bioruptor water bath (Diagenode). After centrifugation the sheared 200 – 500 bp chromatin fragments were diluted with 2x volume of immunoprecipitation buffer II (25 mM Tris, pH 8.0, 150mM NaCl, 2 mM EDTA, pH 8.0, 1 % TritonX100, 7.5 % glycerol), 5 – 10% of input chromatin was saved and the precipitation was carried out with remaining chromatin for 2 - 4 hours at 4 °C using 0.2 - 2 ?g of specific antibody (H3K9ac: Abcam ab4441 and Millipore ABE18, H3K27ac: Abcam ab4729, H3K4me3: Millipore 04-745, H3K27me3: Abcam ab6002) coupled to 15 ?l protein G Dynabeads (Dynal). Beads were washed with low salt buffer (20 mM Tris 1 M, pH 8.0, 150 mM NaCl, 2 mM EDTA, pH 8.0, 1 % TritonX100, 0.1 % SDS), high salt buffer (20 mM Tris, pH 8.0, 500 mM NaCl, 2 mM EDTA, pH 8.0, 1 % TritonX100, 0.1 % SDS), LiCl buffer (10 mM Tris, pH 8.0, 250 mM lithium chloride, 1 mM EDTA, pH 8.0, 0.5 % NP40, 0.5 % sodium-deoxycholate) and TE pH 8.0 containing 50 mM sodium chloride. The immune complexes were eluted in 100 ?l elution buffer (100 mM NaHCO3, 1 % SDS) and, after adding 5 ?l of 5M sodium chloride and proteinase K, the crosslinks were reversed at 65°C overnight. DNA was extracted by using the Ampure PCR purification kit and ChIP quality was validated by realtime PCR.
The libraries were prepared according to Illumina TruSeq DNA Sample Prep Kit
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| 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 and the reads from the SOLiD sequencer were mapped using SHRiMP. The resulting alignment files were used to generate density maps using HOMER.
Genome_build: mm10
Supplementary_files_format_and_content: fastq (unmapped reads), solid_native_csfasta and solid_native_qual files (unmapped reads), and bigWig (density profile of mapped reads)
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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) |
| GSE69096 |
Comprehensive Epigenomic Analysis Reveals Dynamic Regulatory Programs Of Blood Development (ChIP-seq) |
| GSE69101 |
Comprehensive Epigenomic Analysis Reveals Dynamic Regulatory Programs Of Blood Development |
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| Relations |
| BioSample |
SAMN03703188 |
| SRA |
SRX1034737 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1692796_HE_H3K27ME3.bw |
551.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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