GSM5381165: s16_H3K9_WT_30; Saccharomyces cerevisiae; ChIP-Seq
ChIP was performed as described previously (Cai et al., 2011). Briefly, 100 OD600 units of cells were fixed in 1% formaldehyde for 15 min and then quenched in 125 mM glycine for 10 min. 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 µM leupeptin, 5 µM pepstatin A, and Roche protease inhibitor cocktail) and lysed by bead-beating. Lysates were split to two tubes and sonicated for 16 cycles (30 sec on, 1 min off, high output) using the Bioruptor (Diagenode). The lysates were then cleared by two consecutive spins at 15,000 rpm for 10 min. For HA ChIP, lysate was incubated overnight with 10 µg antibody. For histone ChIP, lysate was diluted at 1:10 and incubated overnight with 2 µg antibody. 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 RNase A was then added and incubated for 2 h at 37°C. DNA was then purified using the Qiagen PCR purification kit. ChIP samples were prepared as described above in two biological replicates. We constructed sequencing libraries using KAPA Hyper Prep Kit from Kapa Biosystems. The quality and quantity of constructed libraries were determined using Agilent D1000 ScreenTape System and Invitrogen Qubit Fluorometer. The sequencing was performed by the UTSW Next Generation Sequencing Core Facility using the Illumina NextSeq 500 System.