GSM9365111: GCN5b-KD-HA-PHD1-Myc_Myc-antibody_IAA; Toxoplasma gondii; ChIP-Seq
Crosslinked chromatin was lysed in ice-cold lysis buffer (50mM HEPES KOH pH7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5%NP-40, 0.125% triton X-100, protease inhibitor cocktail) and sheared in shearing buffer (1mM EDTA pH8.0, 0.5mM EGTA pH8.0, 10mM Tris pH8.0, protease inhibitor cocktail) by sonication using a Diagenode Biorupter. Samples were sonicated, for 16 cycles (30 seconds ON and 30 seconds OFF), to 200-500 base-pair average size. Immunoprecipitation was carried out using sheared chromatin, 5% BSA, protease inhibitor cocktail, 10% triton X-100, 10% deoxycholate, DiaMag Protein A-coated magnetic beads (Diagenode) and antibodies (H4K5acK8acK12acK16ac, H4K31me1, HDAC3, and HA). A rabbit IgG antiserum was used as a control mock. After overnight incubation at 4°C on rotating wheel, chromatin-antibody complexes were washed and eluted from beads by using iDeal ChIP-seq kit for Histones (Diagenode) according to the manufacturer's protocol. Samples were decrosslinked by heating for 4 hours at 65°C. DNA was purified by using IPure kit (Diagenode) and quantified by using Qubit Assays (Thermo Fisher Scientific) according to the manufacturer's protocol. For ChIP-seq, purified DNA was used to prepare libraries and then sequenced by Arraystar (USA, http://www.arraystar.com/). ChIP-Sequencing library preparation was performed according to Illumina's protocol Preparing Samples for ChIP Sequencing of DNA. Library Preparation: 10 ng DNA of each sample was converted to phosphorylated blunt-ended with T4 DNA polymerase, Klenow polymerase and T4 polymerase (NEB); An 'A' base was added to the 3' end of the blunt phosphorylated DNA fragments using the polymerase activity of Klenow (exo minus) polymerase (NEB); Illumina's genomic adapters were ligated to the A tailed DNA fragments; PCR amplification was performed to enrich ligated fragments using Phusion High Fidelity PCR Master Mix with HF Buffer (Finnzymes Oy). The enriched product of ~200-700 bp was cut out from gel and purified. Sequencing:  The library was denatured with 0.1M NaOH to generate single-stranded DNA molecules, and loaded onto channels of the flow cell at 8pM concentration, amplified in situ using TruSeq Rapid SR cluster kit (#GD-402-4001, Illumina). Sequencing was carried out by running 100 cycles on Illumina HiSeq 4000 according to the manufacturer's instructions.