Study of Cell-Type-Specific Chromatin Organization: In Situ Hi-C Library Preparation for Low-Input Plant Materials

The three-dimensional folding of chromatin contributes to the control of genome functions in eukaryotes, including transcription, replication, chromosome segregation, and DNA repair. In recent decades, many cytological and molecular methods have provided profound structural insights into the hierarchical organization of plant chromatin. With the Hi-C (high-throughput chromosome conformation capture) technique, analyses of global chromatin organization in plants indicate considerable differences across species. However, our knowledge of how chromatin organization at a local level is connected to tissue-specific gene expression is rather limited. This problem can be tackled by performing fluorescence-activated sorting of fixed nuclei followed by Hi-C, which is tailored for a limited number of input nuclei. Here, we describe an approach of isolating Arabidopsis thaliana nuclei with defined endopolyploidy level and subsequent in situ Hi-C library preparation for low-input plant materials. In principle, this method can be applied to any types of fluorescence-labeled nuclei, offering researchers a useful tool to unveil temporal and spatial chromatin dynamics in 3D in a tissue-specific context.