González-García Mary-Paz, Bustillo-Avendaño Estéfano, Sanchez-Corrionero Alvaro, Del Pozo Juan C, Moreno-Risueno Miguel A
Centro de Biotecnología y Genómica de Plantas (Universidad Politécnica de Madrid-Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria), Campus de Montegancedo, Pozuelo de Alarcón, 28223 Madrid, Spain.
Plants (Basel). 2020 Apr 14;9(4):499. doi: 10.3390/plants9040499.
Fluorescence-activated cell sorting (FACS) is a technique used to isolate specific cell populations based on characteristics detected by flow cytometry. FACS has been broadly used in transcriptomic analyses of individual cell types during development or under different environmental conditions. Different protoplast extraction protocols are available for plant roots; however, they were designed for accessible cell populations, which normally were grown in the presence of light, a non-natural and stressful environment for roots. Here, we report a protocol using FACS to isolate root protoplasts from Arabidopsis green fluorescent protein (GFP)-marked lines using the minimum number of enzymes necessary for an optimal yield, and with the root system grown in darkness in the D-Root device. This device mimics natural conditions as the shoot grows in the presence of light while the roots grow in darkness. In addition, we optimized this protocol for specific patterns of scarce cell types inside more differentiated tissues using the mCherry fluorescent protein. We provide detailed experimental protocols for effective protoplasting, subsequent purification through FACS, and RNA extraction. Using this RNA, we generated cDNA and sequencing libraries, proving that our methods can be used for genome-wide transcriptomic analyses of any cell-type from roots grown in darkness.
荧光激活细胞分选（FACS）是一种用于根据流式细胞术检测到的特征分离特定细胞群体的技术。FACS已广泛应用于发育过程中或不同环境条件下单个细胞类型的转录组分析。针对植物根系有不同的原生质体提取方案；然而，它们是为易于获取的细胞群体设计的，这些细胞群体通常在光照条件下生长，而光照对根系来说是一种非自然且有压力的环境。在此，我们报告一种方案，即使用FACS从拟南芥绿色荧光蛋白（GFP）标记系中分离根原生质体，该方案使用实现最佳产量所需的最少酶量，并且根系在D-Root装置中黑暗条件下生长。该装置模拟自然条件，地上部分在光照下生长而根系在黑暗中生长。此外，我们使用mCherry荧光蛋白针对更分化组织内稀缺细胞类型的特定模式优化了该方案。我们提供了有效原生质体形成、随后通过FACS纯化以及RNA提取的详细实验方案。利用这种RNA，我们生成了cDNA和测序文库，证明我们的方法可用于对在黑暗中生长的根系的任何细胞类型进行全基因组转录组分析。
