An Efficient and Universal Protoplast Isolation Protocol Suitable for Transient Gene Expression Analysis and Single-Cell RNA Sequencing
An Efficient and Universal Protoplast Isolation Protocol Suitable for Transient Gene Expression Analysis and Single-Cell RNA Sequencing
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DOI：
10.3390/ijms23073419
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Wang Yinzheng
中科院分区：
文献类型：
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作者：
Wang Juanjuan;Wang Yang;Lü Tianfeng;Yang Xia;Liu Jing;Dong Yang;Wang Yinzheng
The recent advent of single-cell RNA sequencing (scRNA-seq) has enabled access to the.developmental landscape of a complex organ by monitoring the differentiation trajectory of every.specialized cell type at the single-cell level. A main challenge in this endeavor is dissociating plant.cells from the rigid cell walls and some species are recalcitrant to such cellular isolation. Here, we.describe the establishment of a simple and efficient protocol for protoplast preparation in Chirita.pumila, which includes two consecutive digestion processes with different enzymatic buffers. Using.this protocol, we generated viable cell suspensions suitable for an array of expression analyses,.including scRNA-seq. The universal application of this protocol was further tested by successfully.isolating high-quality protoplasts from multiple organs (petals, fruits, tuberous roots, and hypocotyls).from representative species on the key branches of the angiosperm lineage. This work provides a.robust method in plant science, overcoming barriers to isolating protoplasts in diverse plant species.and opens a new avenue to study cell type specification, tissue function, and organ diversification.in plants.
