Transcriptomics of Arabidopsis sperm cells at single-cell resolution
We present a detailed protocol for isolation of single sperm cells and transcriptome analysis to study variation in gene expression between sperm cells.
Male gametophyte development in flowering plants begins with a microspore mother cell, which upon two consecutive cell divisions forms a mature pollen grain containing a vegetative nucleus and two sperm cells. Pollen development is a highly dynamic process, involving changes at both the transcriptome and epigenome levels of vegetative nuclei and the pair of sperm cells that have their own cytoplasm and nucleus. While the overall transcriptome of Arabidopsis pollen development is well documented, studies at single-cell level, in particular of sperm cells, are still lacking. Such studies would be essential to understand whether and how the two sperm cells are transcriptionally different, in particular once the pollen tube grows through the transmitting tissue of the pistil. Here we describe a detailed protocol for isolation of single sperm cells from growing pollen tubes and analysis of their transcriptome.
KeywordsArabidopsis thaliana Sperm cell Single-cell RNA-seq Transcriptome Semi in vivo pollen tube growth
The authors would like to acknowledge the Genomics Unit and Bioinformatics Unit of IGC. The Genomics Unit was partially supported by ONEIDA Project (LISBOA-01-0145-FEDER-016417) co-funded by FEEI—”Fundos Europeus Estruturais e de Investimento” from “Programa Operacional Regional Lisboa 2020″ and by national funds from FCT—”Fundação para a Ciência e a Tecnologia”. This work was funded by FCT ERA-CAPS project EVOREPRO (ERA-CAPS-0001-2014). CSM acknowledges a doctoral fellowship from FCT (PD/BD/114362/2016) under the Plants for Life PhD Program. JDB received salary support from FCT through an “Investigador FCT” position.
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Conflict of interest
The authors declare that they have no conflict of interest.
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