Neuronal Histone Modifications Profiling

Cortex homogenate was used for H3K27me3 ChIP. As for H3K9me3, Nuclei were extracted from the cortex of male offspring and neuronal (NeuN +) nuclei were enriched by FANS sorting using MoFlo Astrios EQ cell sorter (Beckman Coulter). Native ChIP was performed as described (Jiang et al. 2017 (link)). Briefly, the chromatin was digested with MNase at 28 ℃ for 10 min to obtain mononucleosomes and incubated with anti-H3K9me3 (Abcam AB8898) or anti-H3K27me3 (Millipore, 07–449) antibody at 4 ℃ overnight. The immunoprecipitated complexes were captured by protein A/G magnetic beads (Thermo Scientific, 88803) and washed with low-salt buffer, high-salt buffer, Lithium Chloride buffer and TE buffer. ChIP DNA was then eluted in elution buffer and incubated with RNase A, followed by proteinase K incubation. Finally, ChIP DNA was purified using SPRI magnetic beads (Beckman, B23318).
For ChIP DNA library preparation, 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). Library DNA was size-selected with SPRI beads and sent to GENEWIZ,China, for deep sequencing with Novaseq set paired-end, 150 bp (PE150).