Nuclei isolation protocol from diverse angiosperm species

• DOI: 10.1101/2022.11.03.515090
• Publication Date: 2022-11-05T07:05:13Z
• Authors: Chenxin Li; Joshua C. Wood; Natalie C. Deans; Anne Frances Jarrell; Dionne Martin; Kathrine Mailloux; Yi-Wen Wang; C. Robin Buell
• Keywords: Isolation

Abstract The ability to generate intact nuclei is crucial the success of a variety genomics experiments, such as Assay for Transposase-Accessible Chromatin using sequencing (ATAC- seq), Cleavage Under Targets and Tagmentation (CUT&Tag), nuclei-based single cell (e.g., ATAC-seq RNA-seq). For plants, presence wall presents significant challenges in isolation from tissues. Here, we report an optimized protocol that can be adapted diverse angiosperm species, including maize, soybean, tomato, potato, wheat, starting fresh or frozen Nuclei release achieved through chopping tissue on ice, where key parameter affecting integrity concentration detergent TritonX-100 buffer. method simple, quick, largely centrifugation-free, which debris removed by serial filtration. Initial filtration performed within 20 min. Fluorescence activated sorting then used final purification remove other organelles plastids. uses 500 mg less plant input typically yields at least 100,000 – 200,000 purified per sample, common amount downstream experiments. Throughout protocol, provide guidelines optimization if performing given species first time.