GSM7925177: H3K27me3_Ctrl_rep1; Mus musculus; ChIP-Seq
Cortex homogenate was used for H3K27me3 ChIP. As for H3K9me3, Nuclei was extracted from the cortex of male offspring and neuronal (NeuN+) nuclei were enriched by FACS sorting using MoFlo Astrios EQ cell sorter (Beckman Counter). nuclei were suspended in the MNase digestion buffer (10 mm Tris, pH 7.5, 4 mm MgCl2, and 1 mM Ca2+) and chromatin was digested with MNase at 28 ℃ for 10 min to obtain mononucleosomes and incubated with H3K9me3 (Abcam AB8898) and H3K27me3 (Millipore, 07-449) antibody at 4 ℃ overnight. The immunoprecipitated complexes were captured by Protein AG Magnetic Beads (Thermo Scientific 88803) and washed with 1 mL low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris, 150 mM NaCl), 1 mL high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris, 500 mM NaCl), 500 uL Lithium Cloride buffer (1% IGEPAL-CA 630, 1% Deoxycholic acid, 1 mM EDTA, 10 mM Tris, 0.25 M LiCl), 1 mL TE buffer (1 mM EDTA, 10 mM Tris). ChIP DNA was eluted in 60 uL Elution buffer (0.1M NaHCO3, 1% SDS) and incubated with RNase A, followed by proteinase K incubation. Finally, ChIP DNA was purified using SPRI magnetic beads (Beckman, B23318). End-repairing (Lucigen Corporation, ER0720) and A-tailing (Lucigen Corporation, KL11101K) was performed and then ChIP DNA was ligated (Lucigen Corporation, LK0750H) with Y-adaptor (Vazyme, N802) and subjected to PCR amplification (Vazyme, N618-01).