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Quantitative Bromodeoxyuridine Immunoprecipitation Analyzed by High-Throughput Sequencing (qBrdU-Seq or QBU)

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Genome Instability

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1672))

Abstract

Incorporation into DNA of nucleoside analogs like 5-bromo-2′-deoxyuridine (BrdU) is a powerful tool for in vivo studies of DNA synthesis during replication and repair. Immunoprecipitation of BrdU-labeled DNA analyzed by DNA sequencing (BrdU-IP-seq) allows for genome-wide, sequence-specific tracking of replication origin and replication fork dynamics under different conditions, such as DNA damage and replication stress, and in mutant strains. We have recently developed a quantitative method for BrdU-IP-seq (qBrdU-seq) involving DNA barcoding to enable quantitative analysis of multiple experimental samples subjected to BrdU-IP-seq. After initial barcoding of multiple, individually BrdU-labeled genomic DNA samples, a pooling strategy is used for all subsequent steps including immunoprecipitation, amplification, and sequencing, which eliminates sample-to-sample variability in these steps. Parallel processing of an aliquot of the pooled input sample provides a direct control for the normalization of the data and yields results that allow quantitative comparisons of the experimental samples. Though developed for the analysis of S. cerevisiae, this method should be directly adaptable to other model systems.

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Acknowledgments

We thank current and previous members of the Aparicio lab for comments on the protocol. Research in the Aparicio lab has been funded by NIH R01 GMS05494.

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Authors and Affiliations

  1. Molecular and Computational Biology Program, University of Southern California, Los Angeles, CA, 90089, USA

    Joanna E. Haye-Bertolozzi & Oscar M. Aparicio

Authors
  1. Joanna E. Haye-Bertolozzi
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  2. Oscar M. Aparicio
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Corresponding author

Correspondence to Oscar M. Aparicio .

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Editors and Affiliations

  1. Dipartimento di Bioscienze, Università degli Studi di Milano, Milano, Milano, Italy

    Marco Muzi-Falconi

  2. Donnelly Centre and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

    Grant W Brown

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Haye-Bertolozzi, J.E., Aparicio, O.M. (2018). Quantitative Bromodeoxyuridine Immunoprecipitation Analyzed by High-Throughput Sequencing (qBrdU-Seq or QBU). In: Muzi-Falconi, M., Brown, G. (eds) Genome Instability. Methods in Molecular Biology, vol 1672. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7306-4_16

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  • DOI: https://doi.org/10.1007/978-1-4939-7306-4_16

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7305-7

  • Online ISBN: 978-1-4939-7306-4

  • eBook Packages: Springer Protocols

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