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.