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| Status |
Public on Dec 14, 2013 |
| Title |
High-resolution mapping of transcription factor binding sites on native chromatin |
| Organisms |
Saccharomyces cerevisiae; Drosophila melanogaster |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) and sequencing is widely used for genome-wide profiling of protein binding, but is limited by low resolution and poor specificity and sensitivity. We have implemented a simple genome-wide ChIP protocol that starts with micrococcal nuclease-digested uncross-linked chromatin followed by affinity purification and paired-end sequencing without size-selection. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of the budding yeast TFs Abf1 and Reb1 achieved near-perfect accuracy, in contrast to other profiling methods, which were much less sensitive and specific. Unlike profiles produced using X-ChIP methods such as ChIP-exo, ORGANIC profiles are not biased toward identifying sites in accessible chromatin and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila GAGA Factor and Pipsqueak. Taken together, these results suggest that ORGANIC profiling outperforms current X-ChIP methodologies for genome-wide profiling of TF binding sites.
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| Overall design |
Chromatin immunoprecipitation of micrococcal nuclease-digested native chromatin followed by paired-end sequencing (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin 'ORGANIC' profiling) of DNA-binding proteins Abf1 and Reb1 from S. cerevisiae and GAGA-binding factor (GAF) and Pipsqueak (Psq) from D. melanogaster S2 cells; and, Sono-seq (paired-end sequencing of formaldehyde cross-linked and sonicated chromatin) of yeast nuclei. Reb1 ORGANIC profiling was performed at three different salt (NaCl) concentrations (80, 150, and 600 mM) and Abf1 ORGANIC profiling was done at two different salt concentrations (80 and 600 mM) to achieve varying levels of stringency. GAF and Psq ORGANIC profiles were determined at 80 mM salt. Two replicates each of Reb1 and Abf1 600 mM ORGANIC experiments, mixed Drosophila S2 cell and S. cerevisiae nuclei Reb1 ORGANIC experiments, yeast Sono-seq, and GAF and Psq ORGANIC experiments were performed. Each S. cerevisiae and mixed S2 cell/yeast ORGANIC profiling experiment included separately sequenced input chromatin and ChIP samples. Total of 24 samples.
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| Contributor(s) |
Kasinathan S, Orsi GA, Zentner GE, Ahmad K, Henikoff S |
| Citation(s) |
24336359 |
| |
| Submission date |
Apr 01, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Jorja Henikoff |
| E-mail(s) |
[email protected]
|
| Phone |
206-667-4850
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| Organization name |
Fred Hutchinson Cancer Research Center
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| Department |
Basic Sciences
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| Lab |
Henikoff
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| Street address |
1100 Fairview AV N, A1-162
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109-1024 |
| Country |
USA |
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| Platforms (3) |
| GPL13304 |
Illumina HiSeq 2000 (Drosophila melanogaster) |
| GPL13821 |
Illumina HiSeq 2000 (Saccharomyces cerevisiae) |
| GPL16939 |
Illumina HiSeq 2000 (Drosophila melanogaster; Saccharomyces cerevisiae) |
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| Samples (24)
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| Relations |
| BioProject |
PRJNA195609 |
| SRA |
SRP020912 |
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