High-resolution mapping of transcription factor binding sites on native chromatin
Orsi, Guillermo A.
Zentner, Gabriel E.
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CitationKasinathan, Sivakanthan, Guillermo A. Orsi, Gabriel E. Zentner, Kami Ahmad, and Steven Henikoff. 2014. “High-resolution mapping of transcription factor binding sites on native chromatin.” Nature methods 11 (2): 203-209. doi:10.1038/nmeth.2766. http://dx.doi.org/10.1038/nmeth.2766.
AbstractSequence-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) is widely used for profiling of TF binding, but is limited by low resolution and poor specificity and sensitivity. We present a simple protocol that starts with micrococcal nuclease-digested uncross-linked chromatin and is followed by affinity purification of TFs and paired-end sequencing. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide highly accurate base-pair resolution maps that are not biased toward 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 melanogaster GAGA Factor and Pipsqueak. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:12785963
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