GSM7890708: Rtt101-ChEC_Gal_30sec_rep1; Saccharomyces cerevisiae; OTHER
For each condition, 50 mL of cultures were harvested at room temperature at 1,500 x g for 30 seconds. Cells were quickly resuspended in 1 mL of Buffer A (15 mM Tris pH 7.5; 80 mM KCl; 0.1 mM EGTA; 0.2 mM spermine; 0.5 mM spermidine; 1 mM PMSF; protease inhibitor cocktail), transferred into 2 mL tubes and pelleted at room temperature at 1,500 x g for 30 seconds. Cells were permeabilized by resuspending in 693 mL of Buffer A supplemented with 7 mL of 10% digitonin and incubated at 30°C with shaking for 5 minutes. An aliquot of 100 mL was separated and kept as a negative control (no MNase digestion).  Then, 3.5 mL of 1 M CaCl2 (5 mM final) was added and tubes were immediately placed at 30°C to start MNase cleavage. An aliquot of 100 mL was taken at 30 seconds and immediately mixed with 100mL of Stop Buffer (400 mM NaCl; 20 mM EDTA; 4 mM EGTA; 0.1% SDS). The procedure was repeated for later timepoints.  Once all timepoints were collected, cells were treated with 4 mL of 20 mg/mL proteinase K and incubated 30 minutes at 55°C. DNA was then extracted by the addition of 200 mL of phenol:chloroform:isoamyl followed by 5 min centrifugation at maximum speed at  room temperature. Then, 150 mL of the aqueous phase was transferred into a new tube, and DNA was precipitated by the addition of 1.5 mL of 20 mg/mL glycogen and 500 mL of 100 % ethanol and incubated at -20°C overnight. Precipitated DNA was centrifuged at maximum speed at 4°C for 10 minutes. DNA pellets were washed with 1 mL of 70% ethanol and air-dried for 5 minutes. Dried pellets were resuspended in 48 mL of 10 mM Tris-HCl pH 8.0 and 2 mL of 10 mg/mL RNase A was added for RNA digestion at 37°C for 30 minutes.  For size selection, 100 mL of AmPure XP beads (Beckman Coulter, cat. A63881) was added and the bead:DNA mixture was incubated at room temperature for 5 minutes. Beads were collected by placing tubes on a magnetic rack, and the supernatant was transferred to a new tube containing 55 mL of 10 mM Tris-HCl pH 8.0 and 4 mL of 5 M NaCl. Another DNA extraction and precipitation was performed as described above.  DNA pellets were washed and resuspended in 50 mL of ultrapure water. The concentration was determined using a QuBit dsDNA high-sensitivity assay (Invitrogen, cat. Q32854). NEBnext Ultra DNA library prep kit for Illumina (NEB, cat. E7370L)