GSM8307176: Input_untagged MeCP2 expressed from an estradiol-inducible promoter in S. cerevisiae grown in CSM-uracil rep 2; Saccharomyces cerevisiae; ChIP-Seq
Cells (450 million S.cerevisiae and 68 million Schizosaccharomyces pombe as spike-in) were resuspended in 0.6 mL of lysis buffer (50 mM Hepes-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate) and lysed by bead beating before centrifugation, then resuspended in 1 mL. Samples were sonicated with a Covaris S220 focused-ultrasonicator (peak power 150 W for 7min with 30s cycling off/on, duty factor 14, 200 cycles/burst). One hundred microlitres of lysate was reserved as input and 750 uL was used for immunoprecipitation. Samples were precleared with protein G Dynabeads™ (ThermoFisher cat# 10003D) for 1 hour, incubated overnight anti-GFP antibody (Abcam cat# ab290), and incubated with protein G beads for 1 hour, all at 4°C. Beads were washed twice with lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 0.5 mM EDTA, 1% triton X-100, 0.1% sodium deoxycholate), twice with lysis buffer with 500 mM NaCl, and twice with LiCl wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.6% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA), before 1 wash with TE (pH 8). Samples were eluted in elution buffer (TE pH 8, 1% sodium dodecyl sulfate, 150 mM NaCl, 5 mM dithiothreitol) three times by incubation at 65°C for ten minutes, with shaking. DNA was end-repaired, A-tailed, and ligated to adapters before 17 cycles of PCR amplification with indexed primers. All DNA purification steps used Solid Phase Reversible Immobilization magnetic beads (Omega, M1378). Samples were pooled, gel-purified (200-600 bp fragments) and sequenced on an Illumina NextSeq.