title: Sequencing Spo11 Oligonucleotides for Mapping Meiotic DNA Double-Strand Breaks in Yeast.
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.
authors.affiliation: Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.|Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.
authors.forename: Isabel
authors.initials: I
authors.lastname: Lam
authors.affiliation: Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.|Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.
authors.forename: Neeman
authors.initials: N
authors.lastname: Mohibullah
authors.affiliation: Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA. s-keeney@ski.mskcc.org.|Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA. s-keeney@ski.mskcc.org.|Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA. s-keeney@ski.mskcc.org.
authors.forename: Scott
authors.initials: S
authors.lastname: Keeney
mesh_terms: D015153:Blotting, Western; D002874:Chromosome Mapping / Q000379:methods*; D053903:DNA Breaks, Double-Stranded; D058645:Denaturing Gradient Gel Electrophoresis / Q000379:methods; D004706:Endodeoxyribonucleases / Q000235:genetics*; D047468:Immunoprecipitation / Q000379:methods; D008540:Meiosis; D009841:Oligonucleotides / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D011786:Quality Control; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D000099257:Meiotic Recombination Protein SPO11
chemical_list: D004706:Endodeoxyribonucleases; D009841:Oligonucleotides; D029701:Saccharomyces cerevisiae Proteins; D000099257:Meiotic Recombination Protein SPO11; C518938:Spo11 protein, S cerevisiae
keywords: DNA double-strand breaks; Genome-wide mapping; Immunoprecipitation; Meiosis; Recombination; Spo11; Spo11 oligos; Yeast