Single-cell RNA-seq of maize meiocytes and pollen grains
Single-cell RNA-seq of maize meiocytes and pollen grains
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DOI：
10.1038/s41596-023-00889-6
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发表时间：
2023-10-02
期刊：
影响因子：
14.8
通讯作者：
Nelms，Brad
中科院分区：
文献类型：
--
作者：
Washburn，Mary;Alaniz-Fabian，Jaime;Nelms，Brad
RNA测序（RNA-seq）提供了关于发育途径和突变表型影响的宝贵知识。传统上，植物生殖细胞很难分离用于基因组学，因为它们很罕见，并且通常深深嵌入体细胞组织中。在这里，我们提出了一个协议，以分离单个玉米减数分裂和花粉粒RNA-seq。我们讨论了如何在显微镜下识别和分离每种样品类型，制备RNA-seq文库并分析数据。该技术与其他方法相比具有几个优势，结合了靶向特定稀有细胞类型的能力，同时利用单细胞RNA-seq解决细胞间异质性。该技术与微量的起始材料（例如，一个花药），使得有可能收集密集的时间进程。此外，发育同步花药保存显微镜，允许分期进行平行表达分析。由精通组织解剖的人员在4-5小时内可收集多达200个细胞，由具有分子生物学和基因组学经验的研究人员在2天内完成文库制备。该协议将促进植物繁殖的研究，为植物育种，遗传学和农业提供至关重要的见解。
RNA-sequencing (RNA-seq) provides invaluable knowledge on developmental pathways and the effects of mutant phenotypes. Plant reproductive cells have traditionally been difficult to isolate for genomics because they are rare and often deeply embedded within somatic tissues. Here, we present a protocol to isolate single maize meiocytes and pollen grains for RNA-seq. We discuss how to identify and isolate each sample type under a microscope, prepare RNA-seq libraries and analyze the data. This technique has several advantages over alternative methods, combining the ability to target specific rare cell types while resolving cell-to-cell heterogeneity with single-cell RNA-seq. The technique is compatible with minute amounts of starting material (e.g., a single anther), making it possible to collect dense time courses. Furthermore, developmentally synchronized anthers are saved for microscopy, allowing staging to be performed in parallel with expression analysis. Up to 200 cells can be collected in 4–5 h by someone proficient in tissue dissection, and library preparation can be completed in 2 d by researchers experienced in molecular biology and genomics. This protocol will facilitate research on plant reproduction, providing insights critical to plant breeding, genetics and agriculture.