GSM5905447: Top1-AID_Top2-AID_OsTIR1_MNase_1_rep2; Saccharomyces cerevisiae; MNase-Seq
Preparation and analysis of mono-nucleosomal DNA was performed as described previously (Donovan et al., 2019; Jonge et al., 2017) with minor modifications. Briefly, cells were grown in  synthetic complete media + 2% galactose from OD 0.3 to OD 0.75 and then either untreated or treated with 500 uM auxin for 60 min. Then, cells were fixed in 1% PFA, washed with 1 M sorbitol, treated with spheroplasting buffer (1M sorbitol, 1 mM β-mercaptoethanol, 10 mg/ml zymolyase 100T (US biological, Z1004.250)) and washed twice with 1 M sorbitol. Spheroplasted cells were treated with 0.01171875 U (low MNase) or 0.1875 U (high MNase) micrococcal nuclease (Sigma-Aldrich, N5386-200UN) in digestion buffer (1 M sorbitol, 50 mM NaCl, 10 mM Tris pH7.4, 5 mM MgCl2, 0.075% NP-40, 1 mM β-mercaptoethanol, 0.5 mM spermidine) at 37°C. After 45 min, reactions were terminated on ice with 25 mM EDTA and 0.5% SDS. Samples were treated with proteinase K for 1 h at 37°C and decrosslinked overnight at 65°C. Digested DNA was extracted with phenol/chloroform (PCI 15:14:1), precipitated with NH4-Ac, and treated with 0.1 mg/ml RNaseA/T1. The extent of digestion was checked on a 3% agarose gel. Sequencing libraries were prepared using the KAPA HTP Library Preparation Kit (07961901001, KAPA Biosystems) using 1 ug of input DNA, 5 uL of 10 uM adapter, double-sided size selection before and after amplification using 10 cycles. Libraries were checked on Bioanalyzer High Sensitivity DNA kit (agilent).