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Status |
Public on Jan 08, 2010 |
Title |
IRF4 bound post IL21, seq 1 |
Sample type |
SRA |
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Source name |
murine CD4+ T
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Organism |
Mus musculus |
Characteristics |
genotype/variation: wildtype antibody: IRF4 stimulation: post IL21
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Treatment protocol |
CD4+ T cells were treated with IL-21 (100ng/ml) for 1 hour.
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Growth protocol |
CD4+ T cells were isolated from mouse spleen using anti-CD4-magnetic beads (Miltenyi). For pre-activation of CD4+ T cells, cells were stimulated with 3 μg/ml plate-bound anti-CD3 and 1 μg/ml soluble anti-CD28 for 3 days, and rested for 24 h in fresh RPMI medium.
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Extracted molecule |
genomic DNA |
Extraction protocol |
Chromatin from approximately 2x10^7 cells were used for each ChIP experiment. End repair for ChIP fragments was performed using PNK and Klenow enzyme, followed by treatment with Taq polymerase. Sequencing adaptors were ligated to the repaired ends and ChIP DNA was amplified using adaptor primers for 17 cycles. Fragments around 200bp were isolated from agarose gel and purified DNA was used directly for cluster generation and sequencing following the manufacturer protocols.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina Genome Analyzer II |
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Description |
Chromatin IP against IRF4
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Data processing |
To align sequence tags, analyze their clustering, identify binding motifs, and display results, new code was developed in C language as extensions of the AceView project (Thierry-Mieg and Thierry-Mieg, 2006). Each Solexa 25-mer tag was mapped on the mouse reference genome (version 37), allowing up to 2 mismatches (single base insertion, deletion, or substitution), and retained only if it had a single best position genome-wide. Each tag was shifted downstream by half the effective length of the sonicated sequenced fragments, measured as the maximum of the genome-wide correlation function between hits to the sense and antisense strands of the chromosomes, and represented by an elementary Gaussian with an area of 1 and s of 100 bp, which dampens the sampling fluctuations and introduces a controlled level of fuzziness and “smoothness”. Summing these elementary Gaussians yields a smoothed tag density. Regions where the density locally exceeds a given threshold are analyzed. The number of tags was measured after subtracting the local tags from the matched IgG experiment. If the tag number exceeded a second threshold, it was scored as a peak. Regions with an IgG peak were considered non-specific and eliminated. The peak height indicates the strength of protein binding. The position of the maximum of the peak (i.e., the maximum of the probability of binding the protein) is expected to coincide with the binding motif. The width covered by the peak (typically 200-600 bp) is the region protected by the protein or protein complex. Often, in large peaks, one can distinguish sub-peaks that are expected to correspond to the binding of multiple proteins. When multiple proteins bind to overlapping sequences, their peaks are clustered to generate a broader composite binding site.
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Submission date |
Jan 08, 2010 |
Last update date |
May 15, 2019 |
Contact name |
Dustin E Schones |
E-mail(s) |
dschones@coh.org
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Organization name |
City of Hope
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Street address |
1500 E Duarte Rd.
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City |
Duarte |
State/province |
CA |
ZIP/Postal code |
91010 |
Country |
USA |
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Platform ID |
GPL9250 |
Series (1) |
GSE19198 |
Analysis of interleukin-21-induced Prdm1 gene regulation reveals functional cooperation of STAT3 and IRF4 TFs |
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Relations |
SRA |
SRX015578 |
BioSample |
SAMN00007371 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
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