GSM8417124: Htz1 WT rep2; Saccharomyces cerevisiae; ChIP-Seq
50 mL cell pellets were resuspended in 750 uL FA lysis buffer (50 mM HEPES-KOH, pH7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate and complete protease inhibitor (Roche) along with 1 ml of 0.5 mm acid washed zirconia beads. Lysis was performed by bead beating for 4 cycles of 3 min on and 7 min off in a Mini-Beadbeater-96 machine (Biospec). Whole cell lysates were centrifuged at 14,000 rpm for 3 min at 4 ˚C. Cytoplasmic supernatants were discarded and nuclear pellets were resuspended in 200 uL FA lysis buffer supplemented with 0.1% SDS and 75 uL 0.1 mm zirconia beads. Nuclear samples were sonicated in a Bioruptor (Diagenode) for 4 cycles of 30 sec on and 30 sec off. Samples were centrifuged at 14,000 rpm for 10 min to collect sonicated chromatin fraction which was predominantly enriched with DNA fragments in a range of 100-500 bp in size (verified on agarose gels). Following antibodies were used according to the manufacturer's instructions to prepare ChIP-exo libraries: anti-FLAG (Sigma, M8823), anti-IgG (Sigma, i5006). Chromatin obtained from 50 mL culture was used for a single ChIP experiment. Appropriate antibodies (3-5 ug per ChIP) were conjugated to protein A/G magnetic Sepharose resin (GE Healthcare Life Sciences) at 4 ˚C for 6-8 hr. Unconjugated antibody was removed, then chromatin sample were immunoprecipitated overnight (12-16 hr) at 4 ˚C. For TAP-tagged samples, 40 uL IgG-Dynabeads slurry was used per sample. Subsequent steps were performed as described previously (Rossi et al., 2018). ChIP-exo libraries were gel purified and were sequenced in a paired-end mode with NextSeq 500 (Read_1 40 cycles, Read_2 36 cycles).