Plant Reprod 32 (1) 55-61 [2019-03-00; online 2018-12-26]
We 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.
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