title: GSM5172240: set1_fmn1 Rpb3 input; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: For ChIP experiments: The formaldehyde-crosslinked cell pellets were transferred to to a 2-ml flat-bottom screw-cap tube containing 300 μl glass beads, and disrupt the cells in a mini beadbeater for 5 min or until 90% of cells are disrupted. Sonicate samples in a cold Bioruptor sonicator bath at maximum output, cycling 30 sec on, 30 sec off, for a total of 30 min. Centrifuge samples for 30 min at 16,000 × g, 4°C, and transfer the supernatant to a new 1.5-ml siliconized microcentrifuge tube.After sonication, solubilized chromatin will stay in the supernatant. Libraries were prepared using the Ovation Ultralow v2 kit (NuGEN, catalog #0344). For ATAC-seq experiements: Spheroplasts were prepared by resuspending 3 OD600 units of cells in 400 ul of sorbitol buffer [1.4 M sorbitol, 40 mM HEPES-KOH (pH 7.5), 0.5 mM MgCl2] containing 0.5 mg/ml Zymolyase®-100T (Nacalai Tesque, Inc.) and 10 mM DTT and incubating at 30°C for 15 min. The spheroplasts were washed twice in sorbitol buffer, resuspended in 300 ul of sorbitol buffer, and then, 10 ul of cell suspension (0.1 OD600 units) was used to incubate with 2.5 ul of Tn5 Transposase (TDE1), reaction buffer (TD) from Nextera™ kit and additional 0.01% digitonin in a final volume of 50 ul at 37°C for 30 min. Transposed DNA was purified using MinElute™ PCR Purification Kit (QIAGEN) and then amplified by PCR with custom primers in Nextera™ DNA CD Indexes kit.