Low-input chromatin profiling in Arabidopsis endosperm using CUT&RUN

Endosperm is an essential seed tissue with a unique epigenetic landscape. During endosperm development, differential epigenetic regulation of the maternal and paternal genomes plays important roles in regulating gene expression, especially at imprinted genes. Profiling the endosperm epigenetic landscape on a genome-wide scale is challenging due to its small size, mode of development, and close association with maternal tissue. Here, we applied a low input chromatin profiling method, CUT&RUN (cleavage under targets and release using nuclease), to profile parental-specific chromatin modifications using low numbers of Arabidopsis endosperm nuclei. We demonstrate that CUT&RUN generates genome-wide H3K27me3 landscapes with high sensitivity, specificity and reproducibility using around 20,000 endosperm nuclei purified by flow cytometry and fluorescence-activated cell sorting. H3K27me3 peaks identified by CUT&RUN and previous ChIP (chromatin immunoprecipitation) approaches were largely overlapping, with some distinctions in heterochromatin. The versatility and simplicity of CUT&RUN makes it a viable alternative to ChIP, which requires greater amounts of starting material, and will enable the study of tissue or even cell-type specific epigenomes in Arabidopsis and other plant species. Col plants were emasculated and pollinated by Ler pollens two days later. Seeds were removed from siliques and nuclei from these seeds were extracted using Partec CyStain UV Precise P and immediately FACS sorted based on DAPI. For test experiments using seed coat and embryo nuclei, seeds were removed from siliques sever days after pollination. 2N, 4N and 8N nuclei were collected for CUT&RUN with anti H3K27me3 antibodies or no antibody. For endosperm CUT&RUN, seeds were removed from siliques four days after pollination (embryo at globular stage). 6N nuclei were collected for CUT&RUN either directly without formaldehyde fixation (Unfixed) or after formaldehyde fixation (Fixed). Anti H3 or H3K27me3 antibodies were used in CUT&RUN to profile H3, H3K27me3 landscapes. Normal rabbit IgG were used to generate negative control data. For leaf CUT&RUN, leaves were taken from 3-week-old Col plants. Leaf nuclei was extracted using Partec CyStain UV Precise P for FACS sorting and 2N, 4N and 8N nuclei were collected for CUT&RUN. Endosperm CUT&RUN sequencing samples are designated Endo_{fixation}_{antibody}_N, where fixation = Unfixed or Fixed; antibody = H3, H3K27me3 or IgG; N = the replicate number (either 1 or 2). Seed coat and embryo CUT&RUN sequencing samples are designated 2N_{antibody}_{nuclei number}_{digestion time} , where antibody= H3K27me3 or noab (no antibody). Leaf CUT&RUN sequencing samples are designated Leaf_{antibody}, where antibody= H3, H3K27me3 or IgG.