title: GSM8101452: hop1-loop2rec8_Hop1_ChIP_rep2; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: For ChIP-seq samples: 25 ml of the meiotic cells were collected at different time points and fixed for 30 min in 1% formaldehyde. For CC-seq samples: Protein-DNA Covalent-Complex Mapping (CC-seq) in yeast followed a method previously described (Gittens et al, 2019; preprint: Brown et al, 2023). Briefly, meiotic cell samples were 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, 0.2% Triton X100, 0.1% 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 / 0.5% SDS). Samples were treated with Proteinase K at 50˚C, and purified by ethanol precipitation. For ChIP-seq, libraries were prepared by PCR amplification with TruSeq adaptors (Illumina) as described in (Sun et al., 2015). For CC-seq, DNA ends were filled and repaired using NEB Ultra II end-repair module, with custom dsDNA adapters ligated sequentially to the sonicated, then blocked, ends with rSAP and then 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 NextSeq1000 2x51 bp). For further information, a detailed protocol is available (preprint: Brown et al, 2023).