GSM9515500: CCseq_MJ907_sae2Drec114-8D_6h_rep1; Saccharomyces cerevisiae; OTHER
Protein-DNA Covalent-Complex Mapping (CC-seq) in yeast followed a method previously described (Gittens 2019). Briefly, meiotic cell samples are chilled and frozen at -20˚C for at least 8 hours, then thawed and spheroplasted (in 1 M sorbitol, 50 mM NaHPO4, 10 mM EDTA, 30 min at 37˚C), fixed in 70% ice-cold ethanol, collected by centrifugation, dried briefly, then lysed in STE (2% SDS, 0.5 M Tris, 10 mM EDTA). Genomic DNA was extracted via Phenol/Chloroform/IAA extraction (25:24:1 ratio) at room temperature, with aqueous material carefully collected, precipitated with ethanol, washed, dried, then resuspended in 1xTE buffer (10 mM Tris/1 mM EDTA). Total genomic DNA was sonicated to <500 bp average length using a Covaris M220 before equilibrating to a final concentration of 0.3 M NaCl, 0.1% TritonX100, 0.05% Sarkosyl. Covalent complexes were enriched on silica columns (Qiagen) via centrifugation, washed with TEN solution (10 mM Tris / 1 mM EDTA / 0.3 M NaCl), before eluted with TES buffer (10 mM Tris / 1 mM EDTA / 1% SDS). Samples were treated with Proteinase K at 50˚C, and purified by ethanol precipitation. DNA ends were filled and repaired using NEB Ultra II end-repair module (NEB #E7645), with adapters ligated sequentially to the sonicated, then blocked, ends with recombinant TDP2 treatment in between these steps to remove the 5-phosphotyrosyl-linked Spo11 peptide. Ampure bead cleanups were used to facilitate sequential reactions. PCR-amplified libraries were quantified on a Bioanalyser and appropriately diluted and multiplexed for deep sequencing (Illumina MiSeq 2x75 bp).