title: GSM1859026: Msn2-ChIP-t3; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: ~50 OD WT cycling cells per time point were collected for ChIP of Msn2 and Msn4. Antibodies are as following: Msn2 (y-300, sc-33631), Msn4 (yE-19, sc-15550). Validation is provided on the manufacturer's website. 2.5 µg primary antibody was used per ChIP experiment. Briefly, cells were first fixed in 1% formaldehyde at 25℃ for 15 min and quenched in 125mM glycine at 25℃ for 10 min. Cells were pelleted and washed twice with TBS buffer before freezing. The frozen pellet was resuspended in 0.5 ml ChIP lysis buffer (50 mM HEPES•KOH pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% deoxycholate (DOC), 0.1% SDS, 1 mM PMSF, 5 µM pepstatin A, Roche protease inhibitor cocktail) and split into two tubes and lysed by bead beating. Lysate were combined and expanded into 1 ml and sonicated for 16 cycles (30 sec on, 1 min off, high output) using a Bioruptor (Diagenode, Denville, NJ). The supernatant of the sonicated lysate was pre-cleared and incubated with 2.5 µg primary antibodies. After incubation overnight, 50 µl protein G magnetic beads (Invitrogen, Grand Island, NY, 10003D) were added and incubated for 1.5 h at 4 ℃. Beads were washed twice with ChIP lysis buffer, twice with DOC buffer (10 mM Tris•Cl pH 8.0, 0.25 M LiCl, 0.5% deoxycholate, 0.5% NP-40, 1 mM EDTA) and twice with TE. 100 µl of TES buffer (TE pH8.0 with 1% SDS, 150 mM NaCl, and 5 mM dithiothreitol) was added to resuspend the beads at 65°C for 20 min. Reverse crosslinking was performed by incubation for 6 h at 65°C. An equal volume of TE containing 1.25 mg/ml proteinase K and 0.4 mg/ml glycogen was added to the samples after reverse crosslinking and samples were incubated for 3 h at 37°C. DNA samples were purified using ChIP DNA Clean & Concentrator (ZYMO RESEARCH, D5205, Irvine, CA). Library construction and sequencing were performed using KAPA Hyper Prep Kit (KAPABIOSYSTEMS, KK8502, Wilmington, MA). Briefly, DNA was end repaired and A-tailed. Barcoded adaptors were ligated and DNA was purified with Agencourt AMPure XP beads (Beckman Coulter, A63880, Indianapolis, IN) and amplified by 12-16 cycles. PCR products were gel-extracted and quantified on an Agilent Bioanalyzer.
attributes.tag: GEO Accession
attributes.value: GSM1859026