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| Status |
Public on May 14, 2015 |
| Title |
Genome-wide analysis of H3.3 dissociation in mouse embryonic stem cells |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
Using Tet-inhibited expression of epitope-tagged H3.3 combined with ChIP-Seq we undertook genome-wide measurements of H3.3 dissociation rates across the ESC genome and examined the relationship between H3.3-nucleosome turnover and ESC-specific transcription factors, chromatin modifiers and epigenetic marks.
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| Overall design |
To measure dissociation rates of H3.3, we utilized a TET-repressible ESC line, ES[MC1R(20)], with the expression cassette integrated at the ROSA26 locus. We transfected MC1R ESCs with HA/FLAG-tagged H3.3 controlled by tetracycline response elements. For ‘TET-OFF’ experiments, ESCs were cultured over several passages (weeks) on feeder cells in the absence of DOX and were subsequently passaged onto feeder-free plates prior to the inhibition of HA-H3.3 expression. To repress HA/FLAG-H3.3 expression we treated cells with 2 μg/ml doxycycline hyclate before crosslinking with formaldehyde at various time points. Measurement of H3.3-HA enrichment over time-course was performed using two replicates of ChIP-seq experiments. As a control, input DNA sequencing was performed for every time point
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| Contributor(s) |
Ha M, Kraushaar D, Zhao K |
| Citation(s) |
25598842 |
| Submission date |
Nov 25, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Misook Ha |
| E-mail(s) |
misook.ha@gmail.com
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| Phone |
7732795900
|
| Organization name |
National Heart Lung Blood Institute
|
| Lab |
Laboratory of Epigenome Biology
|
| Street address |
NIH
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platforms (1) |
| GPL9185 |
Illumina Genome Analyzer (Mus musculus) |
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| Samples (10)
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| Relations |
| BioProject |
PRJNA268514 |
| SRA |
SRP050268 |
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