GSM9753421: Input control hippocampus, SAL-SAL, male; Mus musculus; ChIP-Seq
Brain tissues (PFC, hippocampus, hypothalamus, striatum) were collected by bilateral punch (2.0 mm) and flash-frozen at -80C. Nuclei were isolated in cold extraction buffer using a Dounce tissue grinder, filtered (40 um), and enriched by iodixanol gradient centrifugation. NeuN+ neuronal nuclei were labeled with anti-NeuN Alexa Fluor 488 (MAB377X, EMD Millipore) at 4C for 1 h, then sorted by FACS (BD Biosciences). ~10,000 NeuN+ nuclei per replicate were lysed and digested with MNase (88216, Thermo Fisher) in the presence of CaCl2; reaction quenched with EDTA. Chromatin IP was performed using 0.125 ug anti-H3K27ac antibody (39135, Active Motif) coupled to 5 uL protein A Dynabeads (10001D, Thermo Fisher), following the MOWChIP-seq protocol. ChIP-seq libraries were constructed from purified ChIP DNA (~10,000 NeuN+ nuclei) and input DNA (~2,000 nuclei). PFC libraries used xGen 2S DNA Library Prep Kit (10009877, IDT); hippocampus, hypothalamus, and striatum libraries used ThruPLEX DNA-Seq kit (R400674, Takara Bio). Fragment size verified on TapeStation (Agilent) with High Sensitivity DNA Analysis Kit; primer-dimers removed with SPRIselect beads. Libraries quantified with KAPA Library Quantification Kit (4824, Roche), pooled, and sequenced on an Illumina NovaSeq X Plus platform (paired-end 150 nt, ~15 million reads per library).