GSM9744084: 60min_LexA_sth1_rep2; Saccharomyces cerevisiae; MNase-Seq
For MNase-seq, 1.5 OD660 units of cells were harvested and washed sequentially with 1 mL water and 1 mL 1M sorbitol. The pellet was re-suspended in 0.5mL spheroplasting solution (1 M sorbitol, 0.5 mM 2-mercaptoethanol, 0.18 mg/ml zymolyase) and incubated at room temperature for 7 minutes with gentle inversion. Spheroplasts were collected by centrifugation at 500 x g for 3 min, washed twice with 1 mL of 1 M sorbitol, and resuspended in 200 μL digestion buffer (1 M sorbitol, 50 mM NaCl, 100 mM Tris-Cl pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 1 mM 2-mercaptoethanol, 0.5 mM spermidine, 0.075% NP-40). Samples were incubated at 37°C for 8 min with 1U of MNase (Sigma N3755). Reactions were terminated by adding 20 μL quench buffer (250 mM EDTA and 5% SDS). DNA was purified by phenol–chloroform extraction. MNase and ChIP sequencing libraries were prepared using NEBNext ® Ultra™ II DNA Library Prep Kit for Illumina® (Catalog # E7645L) according to the manufacturer's instructions. The sequencing libraries were sequenced on a NextSeq 2000 machine in 2 x 50 bp paired-end sequencing mode at the Genomics Research Incubator at The Pennsylvania State University.