title: GSM1634088: INPUT_1 #4; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: Chromatin immunoprecipitation experiments were conducted according to Kitada et al. (Kitada et al., 2012), with minor modifications. Briefly, 50 OD of yeast cells are crosslinked using 1% formaldehyde for 15 minutes at room temperature and quenched with glycine 125 mM for 5 minutes at room temperature. After two washes with ice-cold PBS, the cells are resuspended in yeast lysis buffer (with 140 mM NaCl for DNMT3b and RNApolII or 500 mM NaCl for histone post-translational modifications) and the same volume of acid-washed glass beads. We disrupted the cells by vortexing for 5 minutes in a Disruptor Genie at 4°C and incubating in iced-water for 2 minutes. We repeated the cycle an additional five times. We collected the lysate by centrifugation after creating a hole on the bottom of the tube with a 25-G needle. We transferred a fraction of the lysate into a microTube (AFA filter – Covaris) and proceeded with the sonication using the Covaris S2 system according to the following parameters: 14 cycles of 30 sec ON, 30 sec OFF; Duty Cycle=5%; Intensity=5%; Cycles/Burst=200. The sonicated lysate is clarified via centrifugation and 50 μl of the supernatant are incubated over night at 4°C with a specific antibody (Supplementary File 6, Panel D). 10 μl of the clarified lysate are used as input control. The next day, immunoprecipitations are incubated 2 hours at 4°C with Protein A Dynabeads. Each wash is performed twice in the following order: low-salt buffer (50mM HEPES pH 7.5, SDS 0.1%, 1% Triton X-100, 0.1% Deoxycholate, 1mM EDTA, 140mM NaCl), high salt buffer (50mM HEPES pH 7.5, SDS 0.1%, 1% Triton X-100, 0.1% Deoxycholate, 1mM EDTA, 500 mM NaCl), LiCl buffer (10 mM Tris-HCl pH 8, 250 mM LiCl, 5mM EDTA, 1% Triton-X, 0.5% NP-40), TE buffer (100 mM Tris-HCl pH 8, 10 mM EDTA). Elution is performed at 65°C with TE/SDS buffer (100 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS). Tubes containing the eluted immunoprecipitations and input controls (additioned of TE/SDS buffer) are incubated overnight at 65°C to reverse the cross-links. RNase treatment is performed at 37°C for 1 hour, followed by a proteinase K treatment for 1 hour at 60°C. Each reaction is then purified using 1.8 volumes of AMPure XP beads according to manufacturer’s instructions. Libraries were prepared with Ovation Ultralow DR kit (Nugen Technologies) starting from 1 ng of purified DNA according to the protocol.
attributes.tag: GEO Accession
attributes.value: GSM1634088