GSM8140553: MNase-seq ∆sds3 (∆NCU01599) strain: N8193; Neurospora crassa; MNase-Seq
Cells were treated with 2 mg of zymolyase (100T) in a 1 ml solution of spheroplast buffer (1M sorbitol, 50mM Tris pH 7.5, 10mM BME) for 15 minutes while rotating at room temperature. Cells were pelleted at full speed and resuspended in 100 ul MNase digestion buffer (1M sorbitol, 50mM NaCl, 10mM Tris pH 7.5, 5mM MgCl2, 1mM CaCl2, 0.075% (v/v) IGEPAL (Sigma), 0.5mM spermidine and 1mM BME). Exonuclease III (NEB) (30 units) and micrococcal nuclease (Takara) were added to samples and rapidly mixed. Digestion proceeded for 10 minutes at 37oC and was quenched with final concentration of 5mM EGTA and 5mM EDTA (from 50mM/50mM stock). MNase concentration was optimized for each strain to yield ~80-90% mononucleosomes (20 units for N8185, N8186, N8193; 40 units for N3752, N8194; 60 units for 3753). RNase (40 ug) treatment was for 1.5 hours at 42C and proteinase K (200 ug) treatment was for 1 hour at 65 C. 10 ug of gel-purified (minelute, Qiagen) mononucleosome DNA was prepared for high-throughput sequencing using the NEBNext DNA Library Prep Master Mix Set for Illumina (NEB).