GSM8647335: K4me3-PH; Homo sapiens; ChIP-Seq
For H3K4me3 (CST, C42D8, 9751S#) and H3-5HT (Beyotime, AG3850) ChIP, cells were fixed with 1% formaldehyde (Thermo, 28908) for 15 min at room temperature (RT). Cells were then washed with PBS and terminated by the addition of 0.125 M glycine. The pellet was resuspended in 1% SDS lysis buffer after removal of cytosolic proteins with cytosolic buffer (50 mM Tris-HCl pH = 8.0, 0.5% NP-40 and 50 mM NaCl). Chromatin DNA was sheared by sonication to an average size of 300-500 bp. The lysates were incubated with IgG or specific antibodies overnight at 4°C and then incubated with protein A/G magnetic beads (MCE, HY-K0202) for 4 hours. After washing, the eluted protein-DNA complex was reversed by heating at 65°C overnight. The immunoprecipitated DNA was purified on QIAquick Spin Columns (Qiagen, 28106) and high-throughput sequencing was performed Sequencing libraries were prepared using the KAPA DNA HyperPrep Kit and subjected to high-throughput sequencing. Briefly, Chip DNA was end-blunted and added with an 'A' base. Then 200–400 bp fragments are gel-isolated and purified. The library was amplified by 18 cycles of PCR.