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

Nature Protocols volume 11, pages 1586–1598 (2016)Cite this article

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Abstract

This protocol is an extension to: Nat. Protoc. 2, 1467–1478 (2007); doi:10.1038/nprot.2007.148; published online 7 June 2007

The 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.

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Figure 1: TaDa can profile the interaction of any protein with DNA or chromatin in a cell-type-specific manner.
Figure 2: Flowchart for processing of TaDa material, with protocol steps listed.
Figure 3: Sample TapeStation plots showing the expected fragment-size distribution following TaDa.
Figure 4: Example log2 ratio profile of RNA Pol II binding in Drosophila neural stem cells.

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Acknowledgements

This work was funded by a Wellcome Trust Senior Investigator Award (103792), Wellcome Trust Programme Grant (092545) and BBSRC Project Grant (BB/L00786X/1) to A.H.B. A.H.B. acknowledges core funding to the Gurdon Institute from the Wellcome Trust (092096) and CRUK (C6946/A14492).

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Author notes

  1. Owen J Marshall & Tony D Southall

    Present address: Present addresses: Menzies Institute for Medical Research, University of Tasmania, Hobart, Australia (O.J.M.), Department of Life Sciences, Imperial College London, London, UK (T.D.S.).,

  2. Owen J Marshall and Tony D Southall: These authors contributed equally to this work.

Authors and Affiliations

  1. The Gurdon Institute, University of Cambridge, Cambridge, UK

    Owen J Marshall, Tony D Southall, Seth W Cheetham & Andrea H Brand

  2. Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK

    Owen J Marshall, Tony D Southall, Seth W Cheetham & Andrea H Brand

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Contributions

O.J.M., T.D.S., S.W.C. and A.H.B. designed the research and analyzed the data; T.D.S., S.W.C. and O.J.M. developed the protocols; and O.J.M., T.D.S., S.W.C. and A.H.B. wrote the paper.

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Correspondence to Andrea H Brand.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Details of the pUAST-attB-LT3-NDam vector used for cloning Dam-fusion proteins for TaDa

(A) Schematic vector map illustrating the key features of the vector. (B) Sequence details of the linker and MCS regions of the vector; translated codons in frame with Dam are shaded. Vector map created using SnapGene software.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1 (PDF 389 kb)

Supplementary Table 1

Sequencing adaptor oligos (DST purity) (XLSX 10 kb)

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Marshall, O., Southall, T., Cheetham, S. et al. Cell-type-specific profiling of protein–DNA interactions without cell isolation using targeted DamID with next-generation sequencing. Nat Protoc 11, 1586–1598 (2016). https://doi.org/10.1038/nprot.2016.084

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