title: GSM1551297: mec1 HU 1h from Control Exp; Saccharomyces cerevisiae; OTHER
library_construction_protocol: Agarose plugs containing yeast cells for spheroplasting were prepared as described (Van Brabant et al., 2001 Mol Cell 7, 705-713). Approximately 10E6 yeast cells from each sample were washed in 50 mM EDTA and embedded in 0.5% Incert low melting agarose (Lonza) in 50 mM EDTA, then cast in a 100-μl mold (BioRad) to solidify.  The plugs were then processed for cell lysis and protein removal as previously described (Feng et al. 2011).  Each processed agarose plug was incubated twice in 5 ml of TE 0.1 buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) for 15 min at room temperature, then incubated twice in 5 ml of 1X End-Repair reaction buffer (33 mM Tris-acetate, pH 7.8, 66 mM potassium acetate, 10 mM magnesium acetate, and 0.5 mM dithiothreitol) for 30 min at room temperature.  Each plug was then mixed with 50 μl of labeling mix (1X End-Repair buffer, 100 μM each of dTTP, dCTP, and dGTP, 84 μM dATP, 16 μM Biotin-14-dATP (Invitrogen), 1 mM ATP, and 3 μl End-It enzyme mix (Epicentre)) and incubated at 25°C for 1.5 h.  The plug was washed twice with 5 ml TE 0.1 buffer at room temperature for 15 min.  The agarose was digested by β-agarase (New England Biolabs) according to manufacturer’s recommendation.  The released genomic DNA was brought to a final volume of 250-300 μl in TE 0.1 buffer and sonicated in a Bioruptor to achieve an average size of smaller than 500 bp.  The fragmented DNA was purified by a Qiagen PCR Cleanup Kit, followed by End-Repair (with unmodified dNTPs) and A-tailing using standard Illumina DNA library construction conditions.   The A-tailed DNA was then brought to a volume of 100 μl with EB buffer (Qiagen) for binding with M270 Dynabeads (Life technologies).  For each DNA sample, 50 μl of bead suspension was first immobilized on a magnet and washed three times each with 100 μl of 1X Binding and Washing buffer (B&W buffer, 5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, pH 8.0, and 1 M NaCl), followed by resuspension in 100 μl of 2X B&W buffer.  The 100-μl DNA sample was then mixed with the beads and incubated at room temperature for 30 min with agitation.  The DNA-bound beads were washed twice each with 100 μl of 1X B&W buffer, then washed twice each with 100 μl of EB buffer.  The washed beads were then resuspended in ligation reaction mix containing 3 μM each of Adapter 1 and Adapter 2 and incubated at room temperature overnight with agitation.   The beads were washed twice each with 200 μl of 1X B&W buffer and twice each with 200 μl of EB and the associated DNA was then amplified by PCR using multiplexed PE PCR primers using standard procedures recommended by Illumina.  The PCR products were purified by AmPure beads to remove free adapters according to manufacturer’s recommendations and the resulting DNA was subjected to 50-bp or 100-bp paired-end sequencing on the Illumina Hi-Seq platform.