GSM8986860: GroupB, mouse 5, antibody H3K4me3, replicate 1; Mus musculus; ChIP-Seq
Adult mice were euthanized by cervical dislocation, and bilateral samples of the frontal cortex (spanning bregma 1.90 to 1.40) were rapidly dissected and flash-frozen at –80°C for later processing. For epigenomic analysis via MOWChIP-seq, the frozen frontal cortex tissue was processed to isolate neuronal nuclei. Fluorescence-activated cell sorting (FACS) was used to enrich for NeuN-positive (NeuN⁺) neuronal populations. Each chromatin immunoprecipitation replicate targeting histone marks H3K4me3 and H3K27ac was performed using approximately 10,000 sorted neuronal nuclei. Chromatin from 10,000 FACS-sorted neuronal nuclei was digested with micrococcal nuclease (MNase) following a published protocol. Chromatin immunoprecipitation was performed using the MOWChIP method (Zhu et al., Nat Protocols, 14, 3366–3394, (2019))with anti-H3K4me3 and anti-H3K27ac antibodies (Active Motif). Libraries were prepared from purified ChIP DNA using the Accel-NGS 2S Plus kit (Swift Biosciences), with experimenters blinded to sample identity and histone mark. Minor protocol modifications were made to optimize PCR amplification and library cleanup. Library were lsequenced on an Illumina HiSeq 4000 (single-end, 50 bp), yielding ~15M reads per ChIP-seq replicate.