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Meiosis pp 51-98 | Cite as

Sequencing Spo11 Oligonucleotides for Mapping Meiotic DNA Double-Strand Breaks in Yeast

  • Isabel Lam
  • Neeman Mohibullah
  • Scott KeeneyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1471)

Abstract

Meiosis is a specialized form of cell division resulting in reproductive cells with a reduced, usually haploid, genome complement. A key step after premeiotic DNA replication is the occurrence of homologous recombination at multiple places throughout the genome, initiated with the formation of DNA double-strand breaks (DSBs) catalyzed by the topoisomerase-like protein Spo11. DSBs are distributed non-randomly in genomes, and understanding the mechanisms that shape this distribution is important for understanding how meiotic recombination influences heredity and genome evolution. Several methods exist for mapping where Spo11 acts. Of these, sequencing of Spo11-associated oligonucleotides (Spo11 oligos) is the most precise, specifying the locations of DNA breaks to the base pair. In this chapter we detail the steps involved in Spo11-oligo mapping in the SK1 strain of budding yeast Saccharomyces cerevisiae, from harvesting cells of highly synchronous meiotic cultures, through preparation of sequencing libraries, to the mapping pipeline used for processing the data.

Key words

Meiosis Recombination DNA double-strand breaks Spo11 Spo11 oligos Genome-wide mapping Yeast Immunoprecipitation 

Notes

Acknowledgements

The Spo11-oligo mapping method was initially developed by Jing Pan and Mariko Sasaki with technical advice from Matthew Neale. Further optimization was done by Xuan Zhu and the authors of this chapter. Nicholas D. Socci developed the sequence mapping pipeline. We are grateful to Julian Lange, Xiaojing Mu, and Sam Tischfield for comments on the manuscript, and Hajime Murakami for suggestions when adapting the protocol to Dynabeads.

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Isabel Lam
    • 1
    • 2
  • Neeman Mohibullah
    • 2
    • 3
  • Scott Keeney
    • 1
    • 2
    • 3
    Email author
  1. 1.Louis V. Gerstner Jr. Graduate School of Biomedical SciencesMemorial Sloan Kettering Cancer CenterNew YorkUSA
  2. 2.Molecular Biology ProgramMemorial Sloan Kettering Cancer CenterNew YorkUSA
  3. 3.Howard Hughes Medical InstituteMemorial Sloan Kettering Cancer CenterNew YorkUSA

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