title: Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing.
abstract: This protocol is an extension to: Nat. Protoc. 2, 1467-1478 (2007); doi:10.1038/nprot.2007.148; published online 7 June 2007The ability to profile transcription and chromatin binding in a cell-type-specific manner is a powerful aid to understanding cell-fate specification and cellular function in multicellular organisms. We recently developed targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling of DNA- and chromatin-binding proteins in vivo without cell isolation. As a protocol extension, this article describes substantial modifications to an existing protocol, and it offers additional applications. TaDa builds upon DamID, a technique for detecting genome-wide DNA-binding profiles of proteins, by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution. TaDa ensures that Dam-fusion proteins are expressed at very low levels, thus avoiding toxicity and potential artifacts from overexpression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to next-generation sequencing technology. TaDa is robust, reproducible and highly sensitive. Compared with other methods for cell-type-specific profiling, the technique requires no cell-sorting, cross-linking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II (Pol II), TaDa can also identify transcribed genes in a cell-type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next-generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure-from collecting tissue samples to generating sequencing libraries-can be accomplished within 5 d.
authors.affiliation: The Gurdon Institute, University of Cambridge, Cambridge, UK.|Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK.
authors.forename: Owen J
authors.initials: OJ
authors.lastname: Marshall
authors.affiliation: The Gurdon Institute, University of Cambridge, Cambridge, UK.|Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK.
authors.forename: Tony D
authors.initials: TD
authors.lastname: Southall
authors.affiliation: The Gurdon Institute, University of Cambridge, Cambridge, UK.|Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK.
authors.forename: Seth W
authors.initials: SW
authors.lastname: Cheetham
authors.affiliation: The Gurdon Institute, University of Cambridge, Cambridge, UK.|Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK.
authors.forename: Andrea H
authors.initials: AH
authors.lastname: Brand
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002843:Chromatin / Q000378:metabolism*; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D019175:DNA Methylation; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D023281:Genomics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D011485:Protein Binding; D017422:Sequence Analysis, DNA / Q000379:methods*; D013997:Time Factors
chemical_list: D002843:Chromatin; D004247:DNA