title: GSM8603569: isw1_null_chd1_null_bio2; Saccharomyces cerevisiae; MNase-Seq
library_name: GSM8603569
library_construction_protocol: Cell pellets were washed in 10 mL water, transferred to 15 mL tubes, and then re-spun for 5 min at 4000 rpm at 4°C. Liquid was removed from cell pellets and then cells were flash frozen in liquid nitrogen. Cell pellets were resuspended in sorbitol-Tris buffer (1 M sorbitol, 50 mM Tris-HCl pH 7.4, 125 mM ß-mercaptoethanol) and then spheroplasted with Zymolyase 100T (US Biological, NC0439195) for 30 min at 30°C while rotating. Spheroplasting efficiency was visually determined under the microscope after mixing 10 µL spheroplasts with 10 µL 10% SDS. Spheroplasted cells were centrifuged at 4000 rpm for 10 min at 4°C. The supernatant was removed, and the pellet was resuspended to 5 mL volume in NP buffer (1 M sorbitol, 50 mM NaCl, 10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.075% NP40, 1 mM ß-mercaptoethanol, and 500 µM spermidine). MNase (500 units; Sigma, #N5386) was resuspended from lyophilized powder in 500 µL Ex50 buffer (10 mM HEPES pH 7.5, 50 mM NaCl, 1.5 mM MgCl2, 0.5mM EGTA pH 8.0, 10% glycerol v/v, 1 mM DTT, and 0.2 mM PMSF) and stored in aliquots at -20°C. Spheroplasted cells in 600 µL aliquots were treated with MNase in a titration ranging from 2 µL to 4.5 µL MNase for 20 min at 37°C with rotation. Then, the reaction was quenched with 100 µL STOP buffer (5% SDS, 100 mM EDTA). Protein was digested and crosslinks were reversed using 30 µL Proteinase K (20 mg/mL; Meridian Bioscience, #BIO-37084) overnight at 65°C.   DNA was purified using equal volume PCI followed by vortexing and centrifugation for 10 min max speed at 4°C in a microcentrifuge. The aqueous layer was transferred to a new tube and thoroughly mixed with equal volume chloroform isoamyl alcohol (24:1 ratio). Samples were centrifuged again for 10 min at 14,000 rpm at 4°C. The aqueous layer was transferred to a new tube and treated with 2 µL glycogen (20 mg/mL), 0.2 M NaCl, and 0.7x-1x volume isopropanol. DNA was precipitated at -20°C for 1 hr, and then centrifuged in a microcentrifuge at maximum speed for 30 min at 4°C. The supernatant was removed and the DNA pellet was washed using 70% ethanol and air dried for 2 mins. The DNA pellet was resuspended in 40 µL 10 mM Tris-HCl pH 7.5. Resuspended DNA was treated with 5 µL RNase A (Thermo Fisher Scientific, EN0531) for 1 hr at 37°C.   To verify MNase digestion, a 15 µL sample of DNA was purified using the E.Z.N.A. Cycle Pure Kit (Omega Bio-Tek, D6492) and separated on a 2% TBE gel. The 3.5 µL MNase-treated condition was chosen for all samples based on the titration series and prepared without DNA size selection. S. pombe MNase-treated chromatin was separately prepared and mononucleosome-sized DNA fragments were prepared by gel isolation and spiked in to S. cerevisiae MNase-digested DNA . Spiked-in MNase-treated DNA was purified using the E.Z.N.A. Cycle Pure kit. Sequencing libraries were prepared with the NEB Next II Ultra DNA library kit (NEB, E7645L) using 50 ng DNA input and 3 PCR cycles. Library quality was verified using fragment analysis (Fragment Analyzer, Advanced Analytical) and submitted for sequencing on an Illumina NextSeq 500 or 2000 instrument for paired-end sequencing to a depth of ~15million uniquely mapped reads per sample. 