title: GSM5537268: CREB ChIP; Mus musculus; ChIP-Seq
library_construction_protocol: Forebrains from four mice each group were mixed and homogenized in PBS containing 2mM EGS (ethylene glycol bis (succinimidyl succinate) and incubated for 20 min at room temperature. Formaldehyde was added to brain homogenates at 1% concentration and incubated for 8 min at room temperature to cross-link chromatin factors and DNA. The cross-linking reaction was stopped by adding glycine to a final concentration of 125 mM and incubated for 5 min at room temperature. Nuclei were pelleted through a sucrose gradient by centrifugation with an SW55Ti rotor (Beckman) at 37,000 rpm for 40 min at 4°C. The isolated nuclei were then subjected to chromatin preparation with Qsonica Q800R3 (at 70% amplitude, 15 s on / 45 s off) for a total of 10 min sonication. Approximately 30 µg sheared chromatin from ABC-CREB or ABC-HDAC4CA mouse forebrains and 4 µg anti-HA antibody (ab9110, Abcam) were applied to a 240 µl reaction system with freshly supplemented protease inhibitor cocktail. Immunoprecipitation was performed strictly according to the ChIP-IT High Sensitivity Kit instructions (53040, Active Motif). Considerable amount anti-HA affinity beads (Smart-lifesciences, SA068001) with sheared chromatin from ABC-mCherry mouse forebrains was seted as IgG ChIP control. Eluted DNA was analyzed with high-throughput sequencing. Libraries were prepared according to Illumina's instructions.