Illumina NovaSeq 6000 paired end sequencing; ChIP-seq to investigate H3K9ac regulation by HDAC3 in trigeminal ganglion of SD rat trigeminal neuralgia model
The brainstem and trigeminal nerve roots on both sides were dissected trigeminal ganglion tissues were incubated with 1 mL IP buffer (20 mM HEPES [pH 7.9], 350 mM NaCl, 0.1% NP-40, 1 mM DTT, 0.2 mM PMSF, 2 mg/ml leupeptin and 2 mg/ml aprotinin) on ice for 30 min, then harvested nucleus by highest speed centrifugation. The supernatant containing nucleus were sonicated to break up the genomic DNA into 200–500 bp fragments. Next, 10% lysates were saved as input and the remaining were incubated with 1 μg IP-grade antibodies of H3K9ac (CST, #9649) and HDAC3 (Abclonal, A19537) at 4 °C overnight. This was followed by incubation for 2 h with Protein A beads at 37 °C to pull down the bound DNA fragments. 3′-dA overhangs were added to the H3K9ac or HDAC3 enriched or input DNA and ligated them to the adapter to build a DNA library.