GSM7659982: Monomac6_H3K27ac_A29_S18; Homo sapiens; ChIP-Seq
Primary AML cells and cell lines were crosslinked with 1% formaldehyde for 10 min at room temperature at a concentration of 30 × 106 cells/mL. The fixed cells were sonicated 10–15 cycles (30 sec on, 30 sec off) to a size of 200–600 bp. Sonicated chromatin was incubated overnight with either 5 µg TP53 antibody (Santa Cruz: DO-1, sc-126X) or 2 µg of antibody against H3K4me3, H3K27me3, or H3K27ac. The next day, antibody–protein complexes were bound to magnetic Protein G beads (Invitrogen, ThermoFisher, Netherlands) for ∼2 hours. Beads were washed with four different wash buffers, and chromatin was eluted from the beads. DNA proteins were de-crosslinked, and samples were purified using the Qiaquick MinElute PCR purification kit. Sequencing samples were prepared according to the manufacturer's protocol (Illumina, Eindhoven, Netherlands). End repair was performed using the precipitated DNA using Klenow and T4 PNK, and subsequently a 3′ protruding A base was generated using Taq polymerase followed by adapter ligation. The DNA was amplified by PCR and ∼300 bp (ChIP fragment + adapters). Samples were sequenced on an Illumina NextSeq 500 system with 2×43 bp paired-end sequencing (PE43).