Protocol | Published:

Using TRIP for genome-wide position effect analysis in cultured cells

Nature Protocols volume 9, pages 12551281 (2014) | Download Citation

  • A Corrigendum to this article was published on 26 March 2015

This article has been updated

Abstract

The influence of local chromatin context on gene expression can be explored by integrating a transcription reporter at different locations in the genome as a sensor. Here we provide a detailed protocol for analyzing thousands of reporters integrated in parallel (TRIP) at a genome-wide level. TRIP is based on tagging each reporter with a unique barcode, which is used for independent reporter expression analysis and integration site mapping. Compared with previous methods for studying position effects, TRIP offers a 100–1,000-fold higher throughput in a faster and less-labor-intensive manner. The entire experimental protocol takes 42 d to complete, with high-throughput sequencing and data analysis requiring an additional 11 d. TRIP was developed by using transcription reporters in mouse embryonic stem (mES) cells, but because of its flexibility the method can be used to probe the influence of chromatin context on a variety of molecular processes in any transfectable cell line.

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  • 16 January 2015

     In the version of this article initially published, the final concentration of tamoxifen used in Step 53 of the Procedure was listed as 1 mM; it should be 1 µM. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank the Netherlands Cancer Institute (NKI) Genomics Core Facility for sequencing support; the NKI Flow Cytometry Facility for fluorescence sorting support; G. Filion, A. Rosado and J.v. Arensbergen for their helpful suggestions; M. Amendola for providing the reference plasmid for IR copy number quantification; and members of our laboratories for their helpful discussions and critical reading of the manuscript. This work was supported by the Netherlands Consortium for Systems Biology (L.F.A.W., M.v.L. and B.v.S.); NWO-ALW open program grant (W.A. and M.v.L.); and EURYI, NWO-ALW VICI and European Research Council advanced grant no. 293662 (B.v.S.).

Author information

Author notes

    • Waseem Akhtar
    •  & Alexey V Pindyurin

    These authors contributed equally to this work.

Affiliations

  1. Division of Molecular Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

    • Waseem Akhtar
    • , Anton Berns
    •  & Maarten van Lohuizen
  2. Division of Gene Regulation, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

    • Alexey V Pindyurin
    • , Ludo Pagie
    •  & Bas van Steensel
  3. Institute of Molecular and Cellular Biology, Siberian Branch of Russian Academy of Sciences, Novosibirsk, Russia.

    • Alexey V Pindyurin
  4. Division of Molecular Carcinogenesis, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

    • Johann de Jong
    • , Jelle ten Hoeve
    •  & Lodewyk F A Wessels
  5. Skoltech Center for Stem Cell Research, Skolkovo Institute for Science and Technology, Skolkovo, Russia.

    • Anton Berns
  6. Delft Bioinformatics Lab, Delft University of Technology, Delft, The Netherlands.

    • Lodewyk F A Wessels
  7. Cancer Genomics Centre, The Netherlands.

    • Maarten van Lohuizen

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Contributions

W.A., A.V.P., M.v.L. and B.v.S. designed and developed the protocol. W.A. and A.V.P. wrote the manuscript. W.A., J.d.J. and L.P. developed the computational pipeline. J.t.H. developed the TRIP web page. M.v.L., B.v.S., L.F.A.W. and A.B. supervised the project and helped in writing.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Waseem Akhtar or Alexey V Pindyurin.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

    Potential artifacts during the preparation of mapping samples for Illumina high-throughput sequencing.

  2. 2.

    Supplementary Figure 2

    Determination of average copy number of IRs in a TRIP pool.

  3. 3.

    Supplementary Figure 3

    Structure of DNA fragments prepared for Illumina high-throughput sequencing.

  4. 4.

    Supplementary Table 1

  5. 5.

    Supplementary Table 2

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DOI

https://doi.org/10.1038/nprot.2014.072

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