ChIP-seq for Histone H3K27Ac in Brain Tissue

ChIP was performed essentially as described in detail in our previous study [37 (link)]. Briefly, brain tissue dissected from 3 animals was pooled, homogenized, and fixed in PBS with 1% formaldehyde for 10 minutes. Nuclei were prepared from the fixed cells and stored at -80°C until use. Thawed nuclei were sonicated using a BiorupterTM UCD-200 (Diagenode, Liège, Belgium) sonicator, and fragmented chromatin was processed for ChIP with 2 ug histone H3K27Ac antibody per sample (Abcam ab4729), using one million nuclei for each IP. IPs were performed in biological replicate, with one pool of 3 samples in each replicate, as previously described. Libraries were prepared from eluted DNA using KAPA LTP library kits (KK8230) using Bioo Scientific index adapters, size-selected using AmpureXP beads (Beckman Coulter, Brea, CA, USA) and quality checked by Qubit 2.0 and Bioanalyzer (Agilent 2100). Samples were sequenced to a depth of 20-30M reads per replicate on an Illumina HiSeq 2500 sequencer using a TruSeq SBS sequencing kit, version 4, in single-end format with fragment length of 100 bp. Base calling and demultiplexing into FASTQ files was done using bcl2fastq v1.8.4 software (Illumina, San Diego, CA, USA).