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

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Title: Cell type-specific profiling of protein-DNA interactions without cell isolation using Targeted DamID with next-generation sequencing
Authors: Southall, TD
Marshall, OJ
Brand, AH
Item Type: Journal Article
Abstract: The ability to profile transcription and chromatin binding in a cell type-specific manner is a powerful approach for 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 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, avoiding toxicity and potential artefacts from over-expression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to Nextgeneration Sequencing technology. TaDa is robust, reproducible, and highly sensitive. Compared to other methods for cell-type specific profiling, the technique requires no cell-sorting, crosslinking 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, 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 days.
Issue Date: 4-Aug-2016
Date of Acceptance: 7-Apr-2016
ISSN: 1750-2799
Publisher: Nature Publishing Group
Start Page: 1586
End Page: 1598
Journal / Book Title: Nature Protocols
Volume: 11
Copyright Statement: Copyright © 2016, Rights Managed by Nature Publishing Group
Keywords: Bioinformatics
Publication Status: Published
Appears in Collections:Faculty of Natural Sciences

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