Chromatin immunoprecipitation and gene expression analysis of neuronal subtypes after fluorescence activated cell sorting.

Abstract

BACKGROUND With advances in cell capture, gene expression can now be studied in neuronal subtypes and single cells; however, studying epigenetic mechanisms that underlie these changes presents challenges. Moreover, chromatin immunoprecipitation (ChIP) protocols optimized for low cell number do not adequately address technical issues and cell loss while preparing tissue for fluorescence activated cell sorting (FACS). Developing a reliable FACS-ChIP protocol without the need for pooling tissue from multiple animals would enable study of epigenetic mechanisms in neuronal subtypes. METHODS FACS was used to isolate dopamine 1 receptor (D1R) expressing cells from the nucleus accumbens (NAc) of a commercially available BAC transgenic mouse strain. D1R+ cells were used to study gene expression as well as histone modifications at gene promoters using a novel native ChIP protocol. RESULTS Isolated cells had enrichment of the dopamine 1 receptor (D1R) mRNA and nearly undetectable levels of GFAP and D2R mRNA. ChIP analysis demonstrated the association of activating or repressive histone modifications with highly expressed or silent gene promoters, respectively. COMPARISON WITH EXISTING METHODS The ChIP protocol developed in this paper enables characterization of histone modifications from ∼30,000 FAC-sorted neurons. CONCLUSIONS We describe a one day FACS-ChIP protocol that can be applied to epigenetic studies of neuronal subtypes without pooling tissue.

DOI: 10.1016/j.jneumeth.2016.02.006