GSM9350754: eaf3Δ, -glucose, Input; Saccharomyces cerevisiae; ChIP-Seq
Cell pellets were washed twice with a buffer containing 100 mM NaCl,10 mM Tris-HCl pH 8.0, 1 mM PMSF, and 1 mM benzamide-HCl, and snap-frozen in liquid nitrogen. Frozen pellets were lysed in 0.45 mL ChIP lysis buffer (50 mM HEPES-KOH pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% deoxycholate, 0.1% SDS,1 mM PMSF, 10 mM leupeptin, 5 mM pepstatin A, and Roche protease inhibitor cocktail) and lysed by bead-beating. Lysates were split into 100ul aliquots for shearing for 40 minutes at default settings- Pulse (N) 50, pulse rate frequency (kHz) 1, burst rate (Hz) 20- using the PIXUL (Active Motif). The lysates were then combined and cleared by two consecutive spins at 15,000 rpm for 10 min. For HA ChIP, the lysate was incubated overnight with 10 µg of antibody. For spike-in normalization, the spike-in chromatin and antibody were added to the reaction as per the manufacturer's recommendation (Active Motif). 25 µl magnetic beads were added and incubated for an additional 1.5 h. Beads were then washed twice with ChIP lysis buffer, once with deoxycholate buffer (10 mM Tris-HCl, 0.25 M LiCl, 0.5% deoxycholate, 1 mM EDTA), and once with TE buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA). Elution was performed using TES buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, and 1% SDS). Reverse crosslinking was performed at 65°C. An equal volume of TE containing 1.25 mg/mL proteinase K and 0.4 mg/mL RNase A was then added and incubated for 2 h at 37°C. DNA was then purified using the QIAGEN PCR purification kit. Library was prepared and sequenced by Novogene.