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Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing

Citation

Marshall, OJ and Southall, TD and Cheetham, SW and Brand, AH, Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing, Nature Protocols, 11, (9) pp. 1586-1598. ISSN 1754-2189 (2016) [Refereed Article]

DOI: doi:10.1038/nprot.2016.084

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.

Item Details

Item Type:Refereed Article
Research Division:Biological Sciences
Research Group:Biochemistry and Cell Biology
Research Field:Biochemistry and Cell Biology not elsewhere classified
Objective Division:Expanding Knowledge
Objective Group:Expanding Knowledge
Objective Field:Expanding Knowledge in the Medical and Health Sciences
Author:Marshall, OJ (Dr Owen Marshall)
ID Code:110965
Year Published:2016
Web of Science® Times Cited:11
Deposited By:Menzies Institute for Medical Research
Deposited On:2016-08-24
Last Modified:2017-11-06
Downloads:0

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