Steponeplus real time pcr

Samples were cut into 2–3 mm 3 pieces, fixed in 1.5% formaldehyde for 10 min, and quenched with glycine. The tissues were mechanically extracted by applying 50–75 strokes using a Dounce homogenizer (Type B). Chromatin was sheared to 200–500 bp using a high-power Bioruptor Plus sonicator for 30 cycles (10 s ON, 10 s OFF). For each ChIP, 1–3 μg of antibodies were conjugated with 100 μL Protein G Dynabeads (Thermo Fisher Scientific, Cat. No. 10004D, 5 mL). Antibodies against the histone marks H3K4me3 (ab8580, Abcam), H3K4me1 (ab8895, Abcam), H3K9me3 (ab8898, Abcam), H3K36me3 (ab9050, Abcam), H3K27ac (ab177178, Abcam), and H3K27me3 (Cat. No: 39155, Active Motif) were used for immunoprecipitation. The immunoprecipitated and input DNA was purified with QIAquick Spin Columns (QIAGEN) and then subjected to library preparation using the ThruPLEX DNA-seq 48D Kit (Rubicon Genomics) according to the manufacturer’s instructions. The libraries were inspected with a Qubit fluorometer, Agilent Bioanalyzer 2100 system, and StepOnePlus Real-Time PCR.