The effect of MNase on nucleosome positioning data
S. cerevisiae genomic DNA was isolated from strain BY4741 (MATa his3?1 leu2?0 met15?0 ura3?0 ) using standard methods and RNA was removed by an additional RNAse treatment for 30 min at 37 °C. The pellet was resuspended in TE and passed 10 times through a 25 gauge needle and then 10 times through a 27 gauge needle. The DNA was run on a 22 x 16 cm, 0.8% agarose TAE gel for 7h at 100V at 4 °C. The genomic DNA band was excised from the gel and the DNA was eluded by electrophoresis in a dialysis bag (Spectra/Por Dialysis Membran MWCO: 3,500 18mm, Spectrum Laboratories, Inc). The DNA was precipitated and the pellet was resuspended in 10mM Tris pH 8.0, 1mM CaCl2. The DNA was digested with 6 x 10-3 units micrococcal nuclease (Sigma Chemical Company) per 10 mg genomic DNA at 37 °C for 5 min. The reaction was stopped by the addition of 1 ml 0.5M EDTA. The DNA was precipitated and after resuspension in 10mM Tris pH 8.0, 1mM CaCl2 electrophoresed on a 6% polyacrylamide TBE gel for 30 min at 200 V and a band corresponding to 140 to 170 base pairs was excised. The DNA was eluded. Library preparation and end-sequencing was performed by the NGS service facility at the Max-Planck-Institut für molekulare Genetik.