High intensity ultrasonic processor

ChIP was performed using the Magna ChIP™ A/G One-Day ChIP Kit (Catalog #17-10085; Merck) according to the manufacturer's instructions. Cross-linking of chromatin was achieved by using 1% formaldehyde for 10 min at 37°C and neutralizing with glycine for 5 min at room temperature. All GC cells were washed with cold 1 mL PBS + proteinase inhibitor (1 mM phenylmethanesulfonyl fluoride, 1 mg peptidase, and 1 mg gastrase inhibitor A). The cells were centrifuged at 716 × g for 5 min at 4°C and disrupted using sodium dodecyl sulfate (SDS) lysis buffer (1% SDS, 10 mM ethylenediaminetetraacetic acid and 50 mM Tris-Hcl pH = 8.0). Subsequent sonication at 150 Hz was performed with four sets of 10 s pulse shears on ice using a high-intensity ultrasonic processor (Cole-Parmer). Equal amounts of chromatin were immunoprecipitated with antibodies targeting TWIST1 (1 : 500, #GTX60776, GeneTex) or H3K27ac (1 : 500, #GTX50903, GeneTex) overnight at 4°C with anti-mouse IgG (1 : 500, ab18413; Abcam) as an isotype control. The total chromatin was used as input. Immunoprecipitation products were collected after incubation with magnetic beads. Beads were washed using a magnetic separation rack, and bound chromatin was eluted in ChIP elution buffer with a proteinase K mixer according to the manufacturer's instructions.