The proposed project will examine age-associated changes in gene expression and chromatin structure in primary human cell subsets isolated from peripheral blood. The goal for 2016/2017 is to carry out the analysis for 30 donors spanning the age-range from 20-80 years. Cytopheresis samples will be fractionated by a combination of magnetic bead purification and flow cytometry into 10 cell subsets. RNA isolated from each cell subset and total peripheral blood mononuclear cells (PBMC) will be sequenced to identify age-associated changes and develop algorithms to de-convulute data from PBMC. Chromatin immunoprecipitation (ChIP) and DNAse I hypersensitivity analysis will be carried out using antibodies directed against specific histone modifications and ATAC protocol, respectively. For these studies we will use only PBMC samples and the most abundant cell subsets to start with. Purified RNA and DNA from ChIP samples will be provided to the Johns Hopkins Deep Sequencing and Microarray Core for Hi-Seq analysis. ATAC samples will be converted into libraries at the NIA and provided to the facility for sequencing.

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