REST targets in the cortex of WT mice and Alzheimer's disease 3xTg mice

We profiled REST targets in WT mice, as well as the Alzheimer's disease 3xTg mouse model, to gain insight into the regulation of physiological pathways by REST, as well as the altered REST cistrome in Alzheimer's disease. Overall design: To gain insight into the regulation of physiological pathways by REST, as well as the altered REST cistrome in Alzheimer?s disease, we used a REST C-terminal antibody to profile the REST binding sites in 11-month-old WT and 3xTg mice. Four biological replicates, each comprised of n=4 frozen cortices from 3xTg mice (females, age 11 months) were processed for ChIP-seq (total n=16 3xTg cortices). Similarly, four biological replicates, each comprised of n=4 pooled frozen cortices from WT mice (females, age 11 months) were processed for ChIP-seq (total n=16 WT cortices). The frozen tissue was sent to Active Motif Services (Carlsbad, CA) to be processed for ChIP-Seq. In brief, the tissue was submersed in PBS + 1% formaldehyde, cut into small pieces and incubated at room temperature for 15 minutes. Fixation was stopped by the addition of 0.125 M glycine (final). The tissue pieces were then treated with a TissueTearer and finally spun down and washed 2x in PBS. Chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (25 ug) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 4 ug of antibody against the C-terminal region of REST (generous gift from Gail Mandel, Vollum Institute). Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Illumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. Steps were performed on an automated system (Apollo 342, Wafergen Biosystems/Takara). After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on Illumina?s NextSeq 500 (75 nt reads, single end).