Using Flow Cytometry to Isolate Maize Meiocytes for Next Generation Sequencing: A Time and Labor Efficient Method

Abstract

Meiosis is essential during sexual reproduction to generate haploid gametes. Genomic or epigenomic studies of meiosis in multicellular organisms using next-generation sequencing (NGS) methods have been limited because of the difficulty of collecting thousands to millions of meiocytes. Here, we describe a simple protocol to efficiently isolate maize male meiocytes from formaldehyde-fixed samples for NGS techniques that require chemical crosslinking to preserve complex interactions or chromatin architecture. Anthers at desired meiotic stages are selected, fixed with paraformaldehyde, and disrupted using a homogenizer. Cell walls are digested to produce a cell suspension containing small somatic cells and large individual meiocytes. The meiocyte fraction is enriched by size separation with cell strainers and further purified by flow cytometry. From 400 anthers, we can isolate 20,000 meiocytes at 98% purity in 6 to 8 hours. © 2018 by John Wiley & Sons, Inc.

ZmRAD51C Is Essential for Double-Strand Break Repair and Homologous Recombination in Maize Meiosis

“…Maize ( Zea mays ) is one of the most prevalent crops worldwide and has been used as a model organism for the cytogenetic study for nearly a century [47,48,49,50,51,52,53]. However, none of the five maize RAD51 paralogues have been characterized yet.…”

DNA methylation dynamics during germline development

“…Future innovations in cell/nucleus isolation techniques will power further understanding of germline DNA methylation reprogramming, especially in female germlines where we currently have little data. Protoplast preparation and fluorescence‐activated cell sorting (FACS) have been exploited to isolate plant male germ cells using cell‐type‐specific fluorescent labelling or different cell properties such as cell size, autofluorescence and DNA density ( Slotkin et al, 2009 ; Borges et al, 2012 ; Calarco et al, 2012 ; Ibarra et al, 2012 ; Hsieh et al, 2016 ; Santos et al, 2017 ; Chang et al, 2018 ; Walker et al, 2018 ; He et al, 2019 ; Zheng and Gehring, 2019 ; Buttress et al, 2022 ). For some species or particular cell types, microdissection serves well ( Sprunck et al, 2005 ; Schmidt et al, 2011 ; Park et al, 2016b ; Flores‐Tornero et al, 2019 ; Zhao et al, 2019 ; Jiang et al, 2020 ), especially now only a few cells are required for DNA methylome sequencing ( Smallwood et al, 2014 ).…”

License to Regulate: Noncoding RNA Special Agents in Plant Meiosis and Reproduction

“…This is possible thanks to the rapid improvement in the efficiency of high-throughput sequencing techniques, often utilizing single-cell protocols and techniques for the isolation of super-pure meiocyte fractions ( Chen et al, 2010b ; Dukowic-Schulze et al, 2020 ). New techniques for isolating PMCs are based on microdissection ( Chen and Retzel, 2013 ); however, in the case of grasses, flow cytometry is also successfully used, which significantly improves the throughput ( Chang et al, 2018 ). It is, however, worth noting that while we have overcome the technical issues with PMC isolation in many plant species, we still do not have efficient methods for isolating MMCs.…”

“…This is possible thanks to the rapid improvement in the efficiency of high-throughput sequencing techniques, often utilizing single-cell protocols and techniques for the isolation of super-pure meiocyte fractions (Chen et al, 2010b;Dukowic-Schulze et al, 2020). New techniques for isolating PMCs are based on microdissection (Chen and Retzel, 2013); however, in the case of grasses, flow cytometry is also successfully used, which significantly improves the throughput (Chang et al, 2018). Johnson et al, 2009;Ding et al, 2012a;Zhou et al, 2012;Zhai et al, 2015;Lee et al, 2020;Fan et al, 2016 Johnson et al, 2009;Zhai et al, 2015;Dukowic-Schulze et al, 2016;Fei et al, 2016;Xia et al, 2019;Zhang et al, 2020b Anther development lncRNA and siRNA O. sativa Long-day-specific male-fertility-associated RNA (LDMAR) is necessary for anther and pollen development.…”