Abstract
Key messageWe report the adaptation of the INTACT method for RNA-sequencing in the endosperm and demonstrate its feasibility for allele-specific expression analysis.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.
Highlights
Double fertilization in flowering plants results in the formation of the embryo and the endosperm, a nurturing tissue similar to the placenta in mammals that supports embryo development (Yan et al 2014)
Our data demonstrate that the INTACT method outperforms manual dissection of the endosperm in terms of tissue purity, and allows endosperm-specific transcript enrichment comparable to samples obtained by LCM
Cell type-specific transcriptome studies are of fundamental value to elucidate the development and function of specific cell types
Summary
Double fertilization in flowering plants results in the formation of the embryo and the endosperm, a nurturing tissue similar to the placenta in mammals that supports embryo development (Yan et al 2014). The endosperm is surrounded by multiple maternal sporophytic cell layers, hindering its. A contribution to the special issue ‘Cellular Omics Methods in Plant Reproduction Research’. Moreno-Romero et al 2017). The methodology is based on biotin tagging of cell-specific nuclei, allowing their subsequent purification from the total nuclei pool using streptavidin-coated magnetic beads. The INTACT system requires the co-expression of two components; the first is a synthetic nuclear targeting fusion (NTF) protein composed of the nuclear envelope WPP domain of the Arabidopsis RAN GTPase activating protein 1 (RanGAP1) attached to GFP and to a biotin ligase recognition peptide, which functions as the substrate for the second component, the Escherichia coli biotin ligase (BirA) (Deal and Henikoff 2011)
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