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

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  • Additional Information
    • Source:
      Publisher: Humana Press Country of Publication: United States NLM ID: 9214969 Publication Model: Print Cited Medium: Internet ISSN: 1940-6029 (Electronic) Linking ISSN: 10643745 NLM ISO Abbreviation: Methods Mol Biol Subsets: MEDLINE
    • Publication Information:
      Publication: Totowa, NJ : Humana Press
      Original Publication: Clifton, N.J. : Humana Press,
    • Subject Terms:
      Bromodeoxyuridine* ; High-Throughput Nucleotide Sequencing*/methods ; Immunoprecipitation*/methods; DNA Barcoding, Taxonomic ; DNA Replication ; DNA, Fungal ; Gene Library ; Quality Control ; Yeasts/genetics
    • 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.
    • Grant Information:
      R01 GM065494 United States GM NIGMS NIH HHS
    • Contributed Indexing:
      Keywords: 5-Bromo-2′-deoxyuridine; DNA barcoding; DNA replication; Data normalization between experiments; Nucleoside analog
    • Accession Number:
      0 (DNA, Fungal)
      G34N38R2N1 (Bromodeoxyuridine)
    • Publication Date:
      Date Created: 20171019 Date Completed: 20180606 Latest Revision: 20191008
    • Publication Date:
      20240829
    • Accession Number:
      10.1007/978-1-4939-7306-4_16
    • Accession Number:
      29043627