title: GSM5702086: TAP-Tagged Input, Exit 30 min Replicate 1; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: Cultures were cross-linked with 1% formaldehyde at RT for 10 min. Reaction was stopped by adding 135 mM glycine. Cultures were then pelleted, washed and resuspended in FA-lysis buffer containing protease/phosphatase inhibitors and 10 mM Na-butyrate. Cells were lysed using a 0.5 mm Zirconia/Silica beads (RPI #9834) in a Mini-Beadbeater high energy cell disruptor (BioSpec) at 4°C, for 6 times 60 seconds. Samples were cooled on ice for 2 min in-between beatings. Subsequently, lysates were sonicated for 20 min in BioRuptor (Diagenode), using high setting and 30 seconds ON/OFF cycles. Cell debris was removed by centrifugation at 10,000 rcf for 15 min at 4°C. Sonication efficiency was confirmed for each sample by running on 0.8% agarose gel. Equal aliquots of sonicated lysates were used per immunoprecipitation reaction with 5 ul of H3K9ac antibody (Active Motif 39317), 5 ul H3K14ac antibody (Active Motif 39697), 5 ul of H3K18ac antibody (Active Motif 39755), 4 ul H3K23ac antibody (Millipore 07-355), 5 ul H3K27ac antibody (Abcam ab4729), 5 ul of H4K12ac antibody (Abcam ab46983), 5 ul H4K16ac antibody (Millipore 07-329), 5 ul Acs2 antibody (Thermo MA5-14810) or 8 ul TAP antibody (Invitrogen CAB1001) preconjugated to Protein G Dynabeads (Life Technologies). Ten percent of the chromatin was saved as input DNA. ChIP reactions were incubated overnight at 4 °C with rotation and washed three times in wash buffer. Immunoprecipitated DNA was eluted from the beads, reversed cross-linked and purified together with the input DNA. Exactly 20 ng DNA (either ChIP or input) was used to construct sequencing libraries using the NEBNext Ultra II DNA library preparation kit for Illumina (New England Biolabs; NEB). Libraries were multiplexed using NEBNext Multiplex Oligos for Illumina (dual index primers) and single-end sequenced (75 bp) on the NextSeq 550 platform (Illumina) in accordance with the manufacturer's protocol.