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.
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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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Lam, I., Mohibullah, N., Keeney, S. (2017). Sequencing Spo11 Oligonucleotides for Mapping Meiotic DNA Double-Strand Breaks in Yeast. In: Stuart, D. (eds) Meiosis. Methods in Molecular Biology, vol 1471. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6340-9_3
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DOI: https://doi.org/10.1007/978-1-4939-6340-9_3
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