Preparing Chromatin and RNA from Rare Cell Types with Fluorescence-Activated Nuclear Sorting (FANS).

Abstract

The application of fluorescent tags to generate cell type-specific translational and transcriptional reporter lines is routine in plants, but separation of different cell types for downstream analyses is hampered by the presence of cell walls and tight connections between cells. Enzymatic removal of cell walls induces a wound response, dedifferentiation, or reprogramming of the resulting protoplasts. Their osmotic and mechanical instability and their large size range are challenging for FACS, a flow -sorting procedure based on differential expression of fluorescent tags. In contrast, plant nuclei are relatively robust and easy to isolate. Here, we describe a protocol for fluorescence-activated nuclear sorting (FANS) that allows efficient purification of very few fluorescence-tagged nuclei from a large background of non-labeled tissue. Purified nuclei are suitable for genome, epigenome, transcriptome, or proteome analyses. We describe in detail how to analyze nuclear RNA and DNA methylation from sorted nuclei representing the limited number of stem cells in the shoot apical meristem of Arabidopsis.