alias: GSM1674717
title: GSM1674717: Sth1td nucleosome 1; Saccharomyces cerevisiae; MNase-Seq
library_construction_protocol: Cells (equivalent to 50 ml culture) were suspended in spheroplasting solution (1M sorbitol, 10 mM Tris pH 7.5, 10 mM 2-MercaptoEthanol, protease inhibitors) and incubated with prepared zymolyase (Sigma) at 37C until the optical density of a 1:100 dilution in 1% SDS dropped to 0.01. Spheroplasts were gently harvested by low-speed centrifugation and stored on ice until ready. Spheroplasts were resuspended with MNase Digestion buffer (50 mM Tris pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 0.1% Triton X-100), split into three tubes, and digested with increasing amounts of MNase at 37C for 8 minutes. The amount of MNase was empirically determined to yield a distribution of mono-, di-, and tri-nucleosomes. MNase digestion was stopped by the addition of 0.1X volumes of stop solution (250 mM EDTA. 5% SDS). Proteinase K was added to the samples, incubated at 37C for 2 hours, and then 65C overnight to reverse cross links. Protein was precipitated from the samples by addition of 8M potassium acetate to 1.2M final concentration, cooling on ice, and centrifugation. DNA was recovered by ethanol precipitation. Samples were treated with RNAase and electrophoresed on a 1.2% agarose gel. Mononucleosomal DNA was excised from the sub-sample whose level of digestion generated an appropriate digestion pattern (about 60% mono, 30% di, and 10% tri) and purified using a Qiagen Gel Extraction Kit. Mononucleosome DNA was phosphorylated with T4 kinase (NEB) prior to ligation of adaptors using Illumina Tru-Seq library construction kit according to manufacturers instructions. No other end repair was performed (fill-in, etc). After limited PCR amplification, samples were size selected on agarose gel and fragments corresponding to mononucleosomes were excised and purified.
identifiers.namespace: GEO
identifiers.id: GSM1674717