title: GSM2472801: MNASE DST1D mutant rep 1; Saccharomyces cerevisiae; MNase-Seq
library_construction_protocol: 500 ml of yeast culture growing exponentially were cross-linked by the addition of formaldehyde to a final concentration of 1 %, followed by 15 minutes of incubation at room temperature. For quenching, glycine was added to 2 % and the sample incubated for 5 minutes at room temperature. Cells were washed three times with cold distilled water. Then, they were resuspended in 10 ml of Buffer Z2 (1 M Sorbitol; 50 mM Tris-HCl pH 7.4; 10 mM β-mercaptoethanol). Spheroplasts were prepared by adding 50 μl of Zymolyase 100T (20 μg/ml). Samples were incubated at 30 ºC during approximately 30 minutes until the optical density at 600 nm was around 10 % of the initial. After washing two times the spheroplasts with cold washing buffer I (1 M sorbitol; 20 mM Tris-HCl pH 8; 1 mM EDTA; 150 mM NaCl), they were resuspended in 1,5 ml of buffer II (20 mM Tris-HCl pH 8; 1 mM EDTA; 150 mM NaCl; 0.1 mM PMSF; 0.2 % Triton X-100). Spheroplasts were separated in 5 tubes with 3 ml of MNase buffer (15 mM Tris-HCl pH 8; 50 mM NaCl; 1.4 mM CaCl2; 0.2 mM EGTA; 0.2 mM EDTA) and different amounts of microccocal nuclease ranging from 500 to 10 mU. Samples were incubated for 30 minutes at 37 ºC, and reactions were stopped by adding 100 μl of stop buffer (0.4 M EDTA; 0.3 M Tris-HCl pH 8) and 150 μl of 10 % SDS. Subsequently, the samples were treated with 75 μl of proteinase K (20 mg/ml) for 30 minutes at 37 ºC, and then at 65 ºC for at least 1 hour. After phenol/chloroform/isoamylic alcohol extraction, DNA was ethanol-precipitated, resupended in double-distilled water and treated with 170 µg/ml RNase A for 30 minutes at room temperature. Digested DNA was resolved in 2 % agarose gels. One of the digestions was chosen, with the following criteria: 1) the trinucleosome band was visible, indicating that there was negligible overdigestion; 2) the mononucleosome band was the most intense (see Fig. 1). The band corresponding to mononucleosomes was purified using Quiagen Gel Extraction kit. DNAs were analyzed by real-time quantitative PCR or ultrasequencing. The naked DNA was prepared by treating the cells exactly as before up to the point of the RNase treatment, with the only difference that no MNase was added. From there, naked DNA was divided into four different tubes with MNase buffer and different ammounts of MNase ranging from 15 to 1 mU. The digestion with a range of fragments similar to that of the chosen chromatin sample (see Fig. 1) was electrophoresed in parallel with the chromatin sample. A slice of gel containing those naked DNA fragments with a similar size to the mononucleosome was cut and the DNA purified
attributes.tag: GEO Accession
attributes.value: GSM2472801