Thruplex dna seq 48d kit

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.

For ChIP-seq, sequencing libraries were prepared using ThruPLEX DNA-seq 48D kit (Rubicon Genomics) following standard protocols. Samples with different dual-index barcodes were combined at equal molar ratios and sequenced as pools. Sequencing of library pools was performed on Illumina HiSeq 2000 or NextSeq 500 system according to Illumina standards, with 50-bp or 75-bp dual-end sequencing. Library de-multiplexing was performed following Illumina standards.