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Quantitative assessment of RNA-protein interactions with high-throughput sequencing–RNA affinity profiling

Nature Protocols volume 10, pages 1212–1233 (2015)Cite this article

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Abstract

Because RNA-protein interactions have a central role in a wide array of biological processes, methods that enable a quantitative assessment of these interactions in a high-throughput manner are in great demand. Recently, we developed the high-throughput sequencing–RNA affinity profiling (HiTS-RAP) assay that couples sequencing on an Illumina GAIIx genome analyzer with the quantitative assessment of protein-RNA interactions. This assay is able to analyze interactions between one or possibly several proteins with millions of different RNAs in a single experiment. We have successfully used HiTS-RAP to analyze interactions of the EGFP and negative elongation factor subunit E (NELF-E) proteins with their corresponding canonical and mutant RNA aptamers. Here we provide a detailed protocol for HiTS-RAP that can be completed in about a month (8 d hands-on time). This includes the preparation and testing of recombinant proteins and DNA templates, clustering DNA templates on a flowcell, HiTS and protein binding with a GAIIx instrument, and finally data analysis. We also highlight aspects of HiTS-RAP that can be further improved and points of comparison between HiTS-RAP and two other recently developed methods, quantitative analysis of RNA on a massively parallel array (RNA-MaP) and RNA Bind-n-Seq (RBNS), for quantitative analysis of RNA-protein interactions.

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Figure 1: Schematics of HiTS-RAP.
Figure 2: DNA template and RNA transcript of HiTS-RAP.
Figure 3: Workflow of HiTS-RAP data analysis.
Figure 4: Examples of protein-binding curves from HiTS-RAP.

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Acknowledgements

We thank J.M. Pagano (Cornell University) for providing NELF-E aptamer and protein constructs, and performing NELF-E EMSA experiments; C.B. Burge (Massachusetts Institute of Technology) for the development of the seminal HiTS-FLIP assay and the scripts used for extraction of protein binding data; B. Mohanty (Medical University of South Carolina) for providing vectors containing the tus gene; K. Szeto and D. Shalloway (Cornell University) for advice on data analysis; W. Zipfel and A. Singh (Cornell University) for help in understanding the optics of the GAIIx; A. Rizzi, C.T. Waters and H. Kwak (Cornell University) for bioinformatics advice; the Cornell Sequencing Core Facility for help in running the GAIIx; and H. Craighead (Cornell University) and the members of the Lis laboratory for helpful discussions on experimental design and the manuscript. This work was supported by US National Institutes of Health grants GM090320 and DA030329 to J.T.L.

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Author notes

  1. Abdullah Ozer and Jacob M Tome: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA

    Abdullah Ozer, Jacob M Tome & John T Lis

  2. Molecular Microbial Pathogenesis Unit, Institut Pasteur, Paris, France

    Robin C Friedman

  3. Illumina, San Diego, California, USA

    Dan Gheba & Gary P Schroth

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  1. Abdullah Ozer
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Contributions

A.O. and J.T.L. conceived of HiTS-RAP. A.O., J.M.T., D.G., G.P.S. and J.T.L. designed the HiTS-RAP protocol. D.G. and G.P.S. supplied sequencing reagents and equipment, as well as technical information. A.O. and J.M.T. performed the experiments. J.M.T. wrote the .xml recipe. J.M.T. and R.C.F. wrote the analysis pipeline. A.O., J.M.T. and J.T.L. wrote the paper.

Corresponding authors

Correspondence to Abdullah Ozer or John T Lis.

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Competing interests

D.G. and G.P.S. are employees of Illumina. All other authors declare no competing financial interests.

Supplementary information

Supplementary Tutorial and Software

Supplementary Tutorial 1 and Supplementary Software 1 (PDF 1179 kb)

Supplementary Software 2

extract_intensity_cif.pl (TXT 6 kb)

Supplementary Software 3

ExtractIntensitiesFromCIF_max.py (TXT 1 kb)

Supplementary Software 4

ExtractIntensitiesFromCIF_T.py (TXT 1 kb)

Supplementary Software 5

HiTS_RAP_Pipeline_Final.py (TXT 19 kb)

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Ozer, A., Tome, J., Friedman, R. et al. Quantitative assessment of RNA-protein interactions with high-throughput sequencing–RNA affinity profiling. Nat Protoc 10, 1212–1233 (2015). https://doi.org/10.1038/nprot.2015.074

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