GSM4794780: Ash1-V5_rep3; Saccharomyces cerevisiae; ChIP-Seq
Cells were resuspended in Lysis Buffer (0.1% deoxycholic acid, 1 mM EDTA, 50 mM HEPES pH 7.5, 140 mM NaCl, 1% Triton X-100, protease inhibitors) and lysed with ten 2-min rounds of bead beating with zirconia beads. Lysed cells were collected by centrifugation, washed once with Lysis Buffer and sonicated in fresh Lysis Buffer for 9 rounds of 30 seconds each with a Misonix sonicator, power level 3.5. Sonicated material was centrifuged, and supernatants were used for chromatin immunoprecipitations. Anti-mouse magnetic Dyna beads (Invitrogen) were prepared for immunoprecipitation by blocking with 1 mg/mL BSA overnight, followed by several hours of incubation with mouse anti-V5 antibody (Abcam). Normalized chromatin amounts were added to prepared Dyna beads and incubated overnight at 4°C with rotation. Beads were washed twice with Lysis Buffer, twice with Lysis Buffer supplemented up to 500 mM NaCl, twice with LiCl Wash Buffer (0.5% deoxycholic acid, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 10 mM Tris pH 8.0) and once with TE. Protein-DNA complexes were eluted from the beads with Elution Buffer (50 mM Tris pH 8.0, 1% SDS, 10 mM EDTA) and incubated at 65°C overnight to reverse cross-links. Multiple identical ChIPs from each cell sample replicate were combined and purified using the MinElute PCR purification kit (QIAGEN), per manufacturer's instructions. NEB Next ChIP-Seq with dual index primers