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| Status |
Public on Aug 05, 2014 |
| Title |
DamID Runx1b::Dam 1 |
| Sample type |
SRA |
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| Source name |
Haemogenic Endothelium
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| Organism |
Mus musculus |
| Characteristics |
cell line: iRunx1b::Damko
strain: 129
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| Growth protocol |
ES cell–derived haemogenic endothelium cells were routinely generated by isolating FLK1+ cells from d3.25 embryonic eody differentiations. FLK1+ cells where maintained on gelatin coated plates in liquid blast medium (IMDM supplemented with 10% FCS, P/S, 2mM L-Glutamine, 20μg/ml transferrin, 0.45mM MTG, 25μg/ml AA, 15% D4T conditioned medium, 5ng/ml VEGF and 10ng/ml IL6). After 2 days of liquid blast culture haemogenic endothelial cells where isolated either by sorting for FLK1+ or for the cKIT+ TIE2+ CD41- phenotype.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from snap frozen samples using the Wizard Genomic DNA Purification Kit (Promega) according to manufacturers instructions and DamID samples where prepared as described (Vogel et al. Nat Protoc 2, 1467-1478, 2007)
For DamID-seq (ChIP-seq) standard barcoded fragment libraries were prepared as described in the Applied Biosystems SOLiD 4 Standard Library Preparation Manual (4445673)
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB SOLiD System 3.0 |
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| Description |
FACS: cKIT+ TIE2+ CD41-
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| Data processing |
Sequencing reads were aligned to mm9 genome using SHRiMP2 with default settings. Reads aligning to multiple loci were discarded.
The BioConductor package BayesPeak was used to call peaks from the uniquely aligned reads for control and test samples seperrately, using the default settings. Peaks with a posterior probability > 0.9 were kept for further analysis.
Isolated peaks (that do not overlap with a peak in the control sample) were retained for subsequent analysis. We then identified all instances where one or more peaks from the test and control samples overlapped and extend the boundaries of these regions to contain all contiguous signal (irrespective of whether that came from control or test samples). We then calculated the RPKM (number of reads per kilo base per million reads) in the region from the test sample and the control sample respectively and ranked regions by the difference in RPKM between the test and control samples (i.e. ∆RPKM = RPKM_test - RPKM_control)
We selected the 10% of regions with the greatest difference and filtered further to discard loci where fold change of test to control, i.e. RPKM_test/RPKM_control, < 2. Peaks in the test sample overlapping with these remaining regions were added to the set of isolated peaks for further analysis.
We applied the Poisson model based scan statistic method (Kulldorff, M.Theory and Methods, 1997; 26:1481-1496) to this set of peaks to find peak-enriched regions in the genome. A 2000bp window was scanned along each chromosome at 400bp increments. At each position of the sliding window, we calculated the number of bases within the window corresponding to a peak. This value, together with length of the window, was used to compute the value of the scan statistic. Monte Carlo sampling was used to establish an approximate distribution of the scan statistic under the null hypothesis.
We selected those windows where the p-value of the scan statistic < 0.05 as peak-enriched windows (PEW) and merged contiguous PEWs to generate larger, peak-enriched regions (PERs), and trimmed the 5' and 3' boundaries of each region to remove nucleotides that do not overlap with any peak. Finally, we merged adjacent PERs closer than 300bp.
PERs were mapped to genome annotation using ENSEMBL (v66) via the BioConductor package annmap, and retaining PERs that overlapped, or within 30kb of a gene. Where multiple genes mapped to a single PER, the closest gene was selected in order to generate a PER_to_gene mapping for each test sample
Genome_build: mm9
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| Submission date |
Feb 24, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
michael lie-a-ling |
| E-mail(s) |
Michael.Lie-A-Ling@ics.manchester.ac.uk
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| Organization name |
Cancer Research UK Manchester Institute
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| Department |
Stem Cell Biology
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| Street address |
The University of Manchester | Wilmslow Road
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| City |
manchester |
| ZIP/Postal code |
M204BX |
| Country |
United Kingdom |
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| Platform ID |
GPL9318 |
| Series (2) |
| GSE55308 |
RUNX1 positively regulates a cell adhesion and migration program in murine hemogenic endothelium prior to blood emergence (DamID-seq) |
| GSE55335 |
RUNX1 positively regulates a cell adhesion and migration program in murine hemogenic endothelium prior to blood emergence |
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| Relations |
| BioSample |
SAMN02664697 |
| SRA |
SRX475922 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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