Systematic Methods for Isolating High Purity Nuclei from Ten Important Plants for Omics Interrogation

Abstract

Recent advances in developmental biology have been made possible by using multi-omic studies at single cell resolution. However, progress in plants has been slowed, owing to the tremendous difficulty in protoplast isolation from most plant tissues and/or oversize protoplasts during flow cytometry purification. Surprisingly, rapid innovations in nucleus research have shed light on plant studies in single cell resolution, which necessitates high quality and efficient nucleus isolation. Herein, we present efficient nuclei isolation protocols from the leaves of ten important plants including Arabidopsis, rice, maize, tomato, soybean, banana, grape, citrus, apple, and litchi. We provide a detailed procedure for nucleus isolation, flow cytometry purification, and absolute nucleus number quantification. The nucleus isolation buffer formula of the ten plants tested was optimized, and the results indicated a high nuclei yield. Microscope observations revealed high purity after flow cytometry sorting, and the DNA and RNA quality extract from isolated nuclei were monitored by using the nuclei in cell division cycle and single nucleus RNA sequencing (snRNA-seq) studies, with detailed procedures provided. The findings indicated that nucleus yield and quality meet the requirements of snRNA-seq, cell division cycle, and likely other omic studies. The protocol outlined here makes it feasible to perform plant omic studies at single cell resolution.

Unveiling the epigenetic landscape of plants using flow cytometry approach

“…As a result, they are not the best choice for relative DNA measures, such as determining ploidy levels. Relative DNA measurements are better suited to fluorescent dyes with ATspecificity, particularly 4 0 ,6-diamidino-2-phenylindole (DAPI) [74] and…”

“…As a result, they are not the best choice for relative DNA measures, such as determining ploidy levels. Relative DNA measurements are better suited to fluorescent dyes with AT‐specificity, particularly 4′,6‐diamidino‐2‐phenylindole (DAPI) [74] and Hoechst [75]. These dyes are unaffected by chromatin condensation because they attach to DNA in a sequence‐specific manner.…”

单细胞测序在植物研究中的应用

“…2021年及之后, 除了大 豆 [35] 、草莓 [36] 、茶树 [37] 、木薯 [38] 、白菜 [39] 、杨树 [40] 等农作物以外, 在苜蓿 [41] 、长春花 [42] 、龙眼 [43] 、荆 芥 [44] 等药用植物中也有了研究. 细胞核是细胞中复杂 且高度特化的细胞器, 包含细胞的大部分遗传信息, 对 于一些植物中特定组织来说, 分离单细胞核相对简 单 [45] . 因此, 近两年来植物单细胞核转录组的研究数量 也逐渐增多(13篇), 但涉及到的物种并不多, 主要涉及 到小麦 [46] 、大豆 [35] 、花生 [47] 、杨树 [48] 、拟南芥 [7] 等.…”

“…细胞核分离相比于原生质体制备的优势在于更容易被 分离, 避免了由于酶促解离而导致的转录组变化. 分离 原生质体需要新鲜的样本, 但细胞核分离可用于保存 的冰冻标本等 [45] . 由于只保留了细胞核中转录本信息, 所以在测序结果中, 不同基因snRNA-Seq所占比例低 于scRNA-Seq, 不利于细胞亚型定位 [54] .…”

3D MXene‐Based Flexible Network for High‐Performance Pressure Sensor with a Wide Temperature Range

“…PEI is an amorphous thermoplastic polymer with high‐temperature resistance, excellent tensile strength, and superior dimensional stability, which has already displayed great potential applications in the aerospace field. [ 20,21 ] In addition, the PEI containing ether bonds exhibits great flexibility and processability, which can construct different micro‐ and macrostructures through various physical processes (e.g., solution casting, [ 22,23 ] extrusion molding, [ 24 ] and electrostatic spinning method [ 25,26 ] ). On the other hand, the emerging MXenes nanosheets with metallic conductivity, excellent mechanical strength, and well hydrophilicity, [ 27‐30 ] can withstand a certain low temperature (below −200 °C) [ 31 ] and high temperature (over 400 °C), [ 32 ] which is suitable for construction of wearable pressure sensor with PEI fibrous network applied in the harsh condition.…”