title: GSM1705338: DNase-seq_W303_S_cerevisiae_Sample2; Saccharomyces cerevisiae; DNase-Hypersensitivity
library_construction_protocol: Intact nuclei were prepared as before (Henikoff et al., 2011) with some alterations. Unfixed yeast cells (W303 strain, asynchronous cells grown in YPD) grown to OD600 0.7 were centrifuged at 2000 rpm for 5 min, washed with sterile water and resuspended in 20 mL buffer Z (0.56M sorbitol, 7.4pH 50mM Tris,autoclaved). After resuspension, 14  μL β-Mercaptoethanol and 500 μL of 10mg/mL zymolyase dissolved in buffer Z were added. Samples were incubated on the bench top for 30 min and inverted every few minutes.  Cells were then centrifuged at 1500 rpm for 6 min at 4 °C and resuspended in 2.5 mL modified NP buffer (1M sorbitol, 50mM NaCl, 7.4pH 10mM Tris, 5mM MgCl2) supplemented with 0.5 mM spermidine, 0.007% B-Mercaptoethanol, and 0.075% NP-40. Next, dilutions of DNase I were prepared on ice to test the best digestion conditions. In 1.5 mL tubes, 400 μL of cell mixture from above was added to 12 μL of the following DNase I solutions: 0.03, 0.1, 0.3, and 1 U/μL. Samples were inverted once and incubated at 37 °C for 16 min. After incubation, 100 μL of stop buffer (5% SDS, 50mM EDTA) was added to terminate the reaction.  Proteinase K (0.2 mg/mL) was added to each tube, inverted to mix and placed at 65 °C overnight. The next day, DNA was recovered by phenol extraction and isopropanol precipitation and run on a 0.8% agarose gel for 3 hours at 85 volts to check sample digestion. Sequencing libraries were prepared as previously described (Song and Crawford, 2010)
attributes.tag: GEO Accession
attributes.value: GSM1705338