Chromatin Immuno-Precipitation (ChIP) Assay

Meiotic induction was performed as described above. Cells were fixed with 1% formaldehyde for 15 min, and cell extracts were prepared as described previously (Hayase et al., 2004). Cell extracts was prepared using a bead shocker (5 cycles of 2,500 rpm, 60 s ON/OFF, Yasui Kikai). The extract corresponding to 3.6 × 10⁸ cells in pertinent mitotic and meiotic conditions were subjected to immunoprecipitation (IP) using Protein-A coated magnetic beads pre-coated with anti-HA (F-7) or anti-Rad51. DNA precipitated with the immune complex was purified by phenol/chloroform extraction and ethanol precipitation after protease-K treatment and quantified by real-time quantitative PCR (Chromo 4, Bio-Rad) using SyberGreen system (EvaGreen Supermix, Bio-Rad). The ChIP primers used were 5′-GGGTTTATAGTGGTGCCGTTC and 5′-ATGCAACGAAGCTTCCTGGC for HIS4-LEU2, 5′-ATGCTGAAGTACGTGGTGACGGAT and 5′-CCTCCGCC ACGACCACACTCT for 0.05 kb from HO-DSB, and 5′-GGTGTGCGGAGTAATCATTTGAGG and 5′-TTATAGGAGA CAGTTTTTCCATCAA for SMC1.