Using Flow Cytometry to Isolate Maize Meiocytes for Next Generation Sequencing: A Time and Labor Efficient Method. Issue 2 (24th May 2018)

• Record Type: Journal Article
• Authors: Chang, Pearl; Tseng, Yu‐Fang; Chen, Pao‐Yang; Wang, Chung‐Ju Rachel
• Editors: Birchler, James; Ecker, Joseph; Martin, Cathie R.; Stitt, Mark; Zhou, Jian-Min
• Other Names: Stacey Gary editorInChief.
• Is Part Of: Current protocols in plant biology. Volume 3:Issue 2(2018)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2018-05-24
• Subjects: anther -- chromatin conformation capture -- high‐throughput sequencing -- maize -- meiocyte -- meiosis; Botany -- Laboratory manuals; Plants; Botany; Botany; Laboratory Manual; Periodical; Laboratory manuals; 580.28
• DOI: 10.1002/cppb.20068
• Languages: English
• ISSNs: 2379-8068

Abstract

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.