Abstract
To enable low-cost, high-throughput generation of cistrome and epicistrome maps for any organism, we developed DNA affinity purification sequencing (DAP-seq), a transcription factor (TF)-binding site (TFBS) discovery assay that couples affinity-purified TFs with next-generation sequencing of a genomic DNA library. The method is fast, inexpensive, and more easily scaled than chromatin immunoprecipitation sequencing (ChIP-seq). DNA libraries are constructed using native genomic DNA from any source of interest, preserving cell- and tissue-specific chemical modifications that are known to affect TF binding (such as DNA methylation) and providing increased specificity as compared with in silico predictions based on motifs from methods such as protein-binding microarrays (PBMs) and systematic evolution of ligands by exponential enrichment (SELEX). The resulting DNA library is incubated with an affinity-tagged in vitro-expressed TF, and TF–DNA complexes are purified using magnetic separation of the affinity tag. Bound genomic DNA is eluted from the TF and sequenced using next-generation sequencing. Sequence reads are mapped to a reference genome, identifying genome-wide binding locations for each TF assayed, from which sequence motifs can then be derived. A researcher with molecular biology experience should be able to follow this protocol, processing up to 400 samples per week.
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Acknowledgements
This work was supported by grants from the National Science Foundation (MCB1024999) and the Gordon and Betty Moore Foundation (GBMF3034) to J.R.E., as well as from the National Science Foundation to A.G. (IOS1114484 and IOS1546873). J.R.E. is a Howard Hughes Medical Institute Investigator.
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R.C.O. and J.R.E. designed the original protocol. R.C.O., A.B., S.-s.C.H., M.G., and A.G. modified and updated the protocol to its current state. J.R.N. performed all the sequencing. A.B., M.G., S.-s.C.H., and J.R.E. wrote the manuscript with contributions from all authors.
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Bartlett, A., O'Malley, R., Huang, Ss. et al. Mapping genome-wide transcription-factor binding sites using DAP-seq. Nat Protoc 12, 1659–1672 (2017). https://doi.org/10.1038/nprot.2017.055
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DOI: https://doi.org/10.1038/nprot.2017.055
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