GSM4850571: tp0 rep2 H3K23ac; Saccharomyces cerevisiae; ChIP-Seq
Cell pellets were resuspended in lysis buffer (50mM HEPES pH 7.5, 140mM NaCl, 1mM EDTA, 2% Tritox X-100, 0.2% Na-deoxycholate, 1X roche protease inhibitor cocktail, 1mM PMSF) and bead beat for 25 minutes. Cell lysates were spun down at 15,000g for 30 minutes. The chromatin pellets were re-suspended in 1000 μl of NP-S buffer (0.5 mM spermidine, 1 mM β-ME, 0.075% NP-40, 50 mM NaCl, 10 mM Tris pH 7.4, 5 mM MgCl2, 1 mM CaCl2) and digested with microccocal nuclease at 37°C for 10 minutes to obtain predominantly mono-nucleosomal DNA. Reactions were stopped with the addition of 10 mM EDTA and digested lysates were clarified by centrifugation at 9000g for 10 minutes. To extract insoluble chromatin, pellets were re-suspended in 300 μl of lysis buffer with 0.2% SDS, and sonicated in a Diagenode Bioruptor at high output for 30 seconds on/30 seconds off four cycles. Extracts were then re-clarified by centrifugation at 9000g for 10 minutes, and the supernatant pooled with the pre-existing extract. The buffer composition of the lysate was adjusted to that of the original lysate buffer (50mM HEPES pH 7.5, 140mM NaCl, 1mM EDTA, 2% Tritox X-100, 0.2% Na-deoxycholate, 1X roche protease inhibitor cocktail, 1mM PMSF), and 10% was set aside as input. The supernatant was pre-cleared by incubation with magnetic protein-G Dynabeads (Life Technologies) for 1 hour at 4°C. Pre-cleared lysates were incubated with αHA (Roche, cat no. 12013819001), αH3K4me3 (Abcam, cat no. ab1012), αH3K23ac (Active Motif, cat no. 39131), or αIgG (Millipore, cat no. PP64) antibodies at 4°C overnight. Immune complexes were precipitated by incubation with magnetic protein-G Dynabeads for one hour at 4°C. Beads were washed twice with lysis buffer, high salt wash buffer (50mM HEPES pH 7.5, 640mM NaCl, 1mM EDTA, 2% Tritox X-100, 0.2% Na-deoxycholate), LiCl was buffer (10mM Tris-HCL pH 8.0, 250mM LiCl, 0.6% NP-40, 0.5% Na-deoxycholate, 1mM EDTA), and once with TE. The immunoprecipitated DNA was subjected to Illumina HiSeq paired-end sequencing.  Library construction for the Illumina platform was performed using a custom procedure for paired-end sequencing. Briefly, 2–10 ng of ChIP material was end-repaired and A-tailed before being ligated to TruSeq PE adaptors. In between each reaction, the material was purified using phenol:chloroform:isoamyl alcohol extraction followed by ethanol precipitation. The resulting material was then amplified in the Phusion HF master mix (NEB) using TruSeq PE PCR primer 1.0 and custom indexed multiplexing primers [5′ AAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC 3′, where “NNNNNN” corresponds to unique hexamer barcodes]. PCR amplification was performed as follows: denaturation at 98 °C for 60 s; eight cycles of (98 °C, 30 s; 65 °C, 30 s; 72 °C, 30 s), and a final extension at 72 °C for 5 min. Amplified libraries were purified using 0.8 (vol) Agencourt AMPure XP solid phase reversible immobilization paramagnetic beads and eluted in 10 mM Tris⋅HCl pH 8.5. An aliquot of each library was run on an Agilent High Sensitivity chip to check the size distribution and molarity of the PCR products. Equimolar amounts of indexed, amplified libraries were pooled, and fragments in the 200–600 bp size range were selected on an 8% (wt/vol) Novex TBE PAGE gel (Invitrogen). An aliquot (1 μL) of the library pool was run on an Agilent High Sensitivity chip to confirm proper size selection and measure DNA concentration. The pooled libraries were diluted to 15 nM and their concentration was confirmed using the Quant-iT dsDNA HS assay kit and Qubit fluorometer (Invitrogen).