GSM2577544: mock for CgPho4 ChIP with ScPho2; Saccharomyces cerevisiae; ChIP-Seq
The cross-linked cell sample was resuspended in ~1 mL of ChIP lysis buffer (50 mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate), and mechanically lysed using a mini-Beadbeater-24 (BioSpec) with 0.5 mm Zirconia beads (BioSpec), using the following setting: 12 cycles of 30s bead beating, with 1 min on ice-water bath in between. The lysed sample was fragmented by sonication using Covaris E220 Adaptive Focus system (setting: Duty Factor: 10%; Peak Incident Power: 175; Cycles/Burst: 200; Time:150s). Afterwards, the sample was centrifuged to remove the unsoluable fraction. 5% of the solubalized chromatin was removed at this step to serve as input sample (not subjected to immunoprecipitation, but was reverse cross-linked and subjected to DNA extraction and library preparation). The remaining supernatant containing the solubalized chromatin was subjected to immunoprecipitation. Briefly, the supernatant was incubated with Dynabeads MyOne Streptavidin C1 (Invitrogen) with rotation at 4C overnight. The dynabeads were then washed with each of the following buffers for 2 x 2 minutes: lysis buffer, high salt wash buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate), lithium wash buffer (10 mM Tris/HCl, pH 8.0, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.1% Na-Deoxycholate), and SDS wash buffer (10 mM Tris/HCl, pH 8.0, 1 mM EDTA, 3% SDS). Finally the dynabeads were washed with TE buffer (10 mM Tris/HCl, pH 8.0, 1 mM EDTA) once for 2 minutes. After washing, cross-linking was reversed by incubation of samples at 65C overnight in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.8% SDS. DNA was then purified using the minElute column (Qiagen). ChIP-seq library was prepared using the NEB NextUltraII DNA library prep kit, following the manufacturer's protocol.
GEO Accession
GSM2577544