title: GSM4192347: WT YCP50 HHT2-HHF2 Galactose; Saccharomyces cerevisiae; MNase-Seq
library_construction_protocol: Yeast nuclei was prepared according to (Almer and Horz, 1986). Cells were harvested  and incubated in 2 volume of preincubation solution (0.7 M ß-mercaptoethanol, 28 mM EDTA) for 25-30 mins. After washing with 1M sorbitol, cell wall was digested with Zymolyase. Spheroplasts were washed with 1M sorbitol and lysed with Ficoll buffer (18 % Ficoll, 20 mM KH2PO4 pH 6.8, 1 mM MgCl2, 0.25 mM EGTA, 0.25 mM EDTA). Nuclei was centrifuged and stored at -80 ̊C before use. For MNase digestion, nuclei was washed and dissolved in MNase digestion buffer (15 mM Tris-Cl pH 7.5, 50 mM NaCl, 1.4 mM CaCl2, 0.2 mM EGTA, 0.2 mM EDTA, 5 mM ß-mercaptoethanol). Equal volume of nuclei was digested with increasing amount of MNase for 20 mins at 37 ̊C. Reaction was stopped with 10 mM EDTA, 1% SDS and 50 mM Tris-Cl pH 8.5, followed by Proteinase K incubation for 30 mins at 37 ̊C. DNA was isolated by Phenol:Chloroform extraction and precipitation with 100% ethanol. Pellet was dissolved in TE buffer and RNA was removed with RNAse A incubation at 37 ̊C for 60 mins. DNA was precipitated with isopropanol, washed with 70% ethanol and dissolved in 50 μl TE. 25 μl DNA was loaded on 1.7% agarose gel and resolved for 4 hours at 2 V/cm. Appopriately digested sample with 70% mono-, 20% di- and 10% tri-nucleosome bands were used for library preparation without cutting mononucleosome band. Libraries were prepared from whole MNase digested DNA. DNA fragments longer than 500 bp were removed using ampure size selection. 50 ng of size-selected DNA was used for library preparation with NEBNext Ultra II DNA Library Prep Kit, according to the manufacturer's protocol. For PCR amplification step, 3 or 4 cycles were performed. Quality control on final libraries was performed using DNA 1000 Bioanalyzer and fragment length of ~270-280 bp was consistently observed, corresponding to ~150-160 bp insert length. Fro ATAC-seq, libraries were prepared using Nextera DNA Library Prep Kit in 25 ul reaction. Final fragment size of 100-600 bp were retained during Ampure purification.
attributes.tag: GEO Accession
attributes.value: GSM4192347