Illumina HiSeq 4000 sequencing; ChIP-seq experiment in J20 hAPP mice and Pilocarpine-treated mice using deltaFosB antibody against whole-hippocampal lysate
ChIP-seq experiment in J20 hAPP mice and Pilocarpine-treated mice using deltaFosB antibody against whole-hippocampal lysate
To verify that hippocampal samples used for ΔFosB ChIP-seq came from mice with seizures and high ΔFosB expression, we first performed immunohistochemistry (Cell Signaling, D3S8R) to detect ΔFosB in one hemibrain (right side). We isolated the entire hippocampal formation from each mouse by chilling each hemibrain (left side) on an ice block, and rolling out the entire hippocampal formation using a molt elevator (Roboz, RS-8830). To perform ChIP, after removing 2% of sheared chromatin samples to use as input, chromatin fragments bound to ΔFosB were isolated using a rabbit anti-ΔFosB antibody (Cell Signaling, D3S8R), with normal rabbit IgG (Millipore 12-370) used as a negative control. All antibody concentrations were 2 μg/reaction, and samples were processed using the Magna ChIP A Kit (Millipore) for pulldown. Library preparation of ~300bp-long ChIP-seq sample fragments (NEBNext Kit, New England Biolabs) was performed by the University of Pennsylvania Next-Generation Sequencing Core prior to Illumina sequencing.
Experimental Factor: genotype
non-transgenic J20 littermate
Experimental Factor: compound
none
Experimental Factor: immunoprecipitate
input DNA