Key message

We report the adaptation of the INTACT method for RNA-sequencing in the endosperm and demonstrate its feasibility for allele-specific expression analysis.

Abstract

Tissue-specific transcriptome analyses provide important insights into the developmental programs of defined cell types. The isolation of nuclei tagged in specific cell types (INTACT) is a versatile method that allows to isolate highly pure nuclei from defined tissue types that can be used for several downstream applications. Here, we describe the adaptation of INTACT from endosperm nuclei for high-throughput RNA-sequencing. By analyzing the ratio of parental reads and tissue-specific gene expression in the endosperm, we could assess the contamination level of our samples. Based on this analysis, we estimate that in most of the samples the contamination level is lower than in previously published datasets. We further show that the nuclear transcriptome and total transcriptome of the endosperm are well correlated. Together, our data show that INTACT of the endosperm is a reliable methodology for endosperm-specific transcriptome analysis that overcomes the limitation of time-consuming manual endosperm dissection that is connected with high levels of maternal tissue contamination. INTACT does not rely on expensive equipment and can be set up in every standard molecular biology laboratory, making it the method of choice for future molecular studies of the endosperm.

中文翻译：

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应用 INTACT 系统进行胚乳特异性转录组分析。

 关键信息

我们报告了 INTACT 方法在胚乳 RNA 测序中的应用，并证明了其用于等位基因特异性表达分析的可行性。

 抽象的

组织特异性转录组分析为特定细胞类型的发育程序提供了重要的见解。分离特定细胞类型中标记的细胞核 (INTACT) 是一种多功能方法，可以从特定的组织类型中分离出高纯度的细胞核，可用于多种下游应用。在这里，我们描述了 INTACT 从胚乳核中的应用，以进行高通量 RNA 测序。通过分析胚乳中亲本读数和​​组织特异性基因表达的比率，我们可以评估样品的污染水平。根据此分析，我们估计大多数样本的污染水平低于之前发布的数据集。我们进一步表明，胚乳的核转录组和总转录组具有良好的相关性。总之，我们的数据表明，胚乳的 INTACT 是一种可靠的胚乳特异性转录组分析方法，克服了耗时的手动胚乳解剖的局限性，该局限性与高水平的母体组织污染有关。 INTACT 不依赖昂贵的设备，可以在每个标准分子生物学实验室中设置，使其成为未来胚乳分子研究的首选方法。