ChIP Assay for H3K4me3 Enrichment

The EZ-Magna ChIP A/G kit (17–371; Millipore, Billerica, MA, USA) was applied in accordance with manufacturer’s instructions. After being sonicated, the cells were centrifuged at 12,000×g at 4 °C for 10 min to remove the insoluble components. The cells were then added with Protein G Agarose, incubated at 4 °C for 1 h and centrifuged at 5000×g for 1 min to remove the supernatant. Then 10 µL of supernatant was used as “Input” and the remaining supernatant was divided into two parts, which were further treated with H3K4me3 antibody (ab185637; 1:20, Abcam) and NC rabbit anti-human IgG (ab2410; 1:25, Abcam) at 4 °C using overnight incubation. Protein G Agarose was inverted and incubated at 4 °C for 1 h to precipitate the protein-DNA complexes. Following centrifugation at 5000×g for 1 min, the supernatant was discarded. The non-specific complex (protein-DNA complex) was eluted, and de-crosslinked at 65 °C. The recovered and purified DNA fragments were used as amplification templates for RT-qPCR experiments, and the enrichment of H3K4me3 in the LSD1 promoter region was detected by ChIP assay [10 (link), 12 (link)].