GSM7467003: 1-2-MTX-14 [Break-seq]; Saccharomyces cerevisiae; OTHER
10^8 cells prewashed with 50mM EDTA were embedded into each agarose plug (0.5% Incert agarose in 50mM EDTA).  The cells were then spheroplasted for 4h at 37 °C with gentle shaking, followed by an overnight incubation with LDS. The cells were then washed twice for 30 min each  with 0.2 X NDS and TE pH8.0 for 30 min each. Agarose plugscontaining DNA were washed once with TE pH 8.0, followed by in-gel labeling using biotin tagged dATP.  Plugs containing labelled DNA were then digested with β-agarase to digest the agarose.  The eluted DNA was then sonicated to using Covaris M220 sonicator to get an average fragment size of 300 bp.  The  sonicated DNA was end repaied followed by dA Tailing. The biotinylated, endrepaired and dA Tailed DNA was subjected to IP using M270 Dynabeads to caputed the biotin labeled DNA fragments. The captured DNA was then subjected to library construction by attaching Solexa adaptors to the ends using T4 DNA ligase. 400 ng of the DNA was then subjectedto PCR amplification using KAPA Hotstart Ready Mix (KAPA) using a unique sequencing primer per sample.  The amplified DNA was then purified using AMPure beads to remove free adaptors.  The libraries were then sequenced on Illumina Hi-Seq 2500 platform  (150 bp paired-end reads).