Single‐nucleus RNA sequencing of plant tissues using a nanowell‐based system

Abstract

Single-cell genomics provides unprecedented potential for research on plant development and environmental responses. Here, we introduce a generic procedure for plant nucleus isolation combined with nanowellbased library preparation. Our method enables the transcriptome analysis of thousands of individual plant nuclei. It serves as an alternative to the use of protoplast isolation, which is currently a standard methodology for plant single-cell genomics, although it can be challenging for some plant tissues. We show the applicability of our nucleus isolation method by using different plant materials from different species. The potential of our single-nucleus RNA sequencing method is shown through the characterization of transcriptomes of seedlings and developing flowers from Arabidopsis thaliana. We evaluated the transcriptome dynamics during the early stages of anther development, identified stage-specific activities of transcription factors regulating this process, and predicted potential target genes of these transcription factors. Our nucleus isolation procedure can be applied in different plant species and tissues, thus expanding the toolkit for plant single-cell genomics experiments.

A 3D gene expression atlas of the floral meristem based on spatial reconstruction of single nucleus RNA sequencing data

“…We performed single-nuclei RNA-seq (snRNA-seq) 4 , 7 , where nuclei were collected by fluorescence-activated DAPI-stained nuclei sorting (FANS) after 4 days (stage 5) of DEX-induction. We isolate nuclei instead of protoplasts to avoid the transcriptome changes that protoplast may create 4 , 39 , 40 . After, snRNA-seq datasets were produced using the 10x Chromium system.…”

“…Characterizing gene expression dynamics and heterogeneity at single-cell resolution is essential to understanding the molecular mechanisms underlying cellular differentiation in multicellular organisms. Technologies based on cell dissociation 1 – 3 or nuclei isolation 4 – 7 combined with high-throughput transcriptome sequencing (scRNA-seq/snRNA-seq) allow for the characterization of the transcriptomes of hundreds of thousands cells at single-cell resolution. However, the physical location of these cells is lost during the experimental process.…”

Simultaneous Analysis of Single-nucleus Transcriptome and Chromatin Accessibility Unveils the Mechanisms of Leaf Cell Development in Arachis hypogaea L.

“…One-week old peanut seedlings grown in an incubator with normal conditions (25℃, 12h light-dark alternation), leaf samples were collected and mixed at 3, 5, and 7 days (9 clock, am) after the germination. Leaves nuclei were isolated following a previously published protocol with slight modi cations (Sunaga-Franze et al, 2021;Conde et al, 2021). Approximately 1 g of the fresh leaf was ground into a powder by freezing it with liquid nitrogen.…”

Simultaneous Establishing Single-cell Transcriptome Atlas and Chromatin Accessibility Landscapes in Allotetraploid Leguminous Plant

“…Peanut seedling leaf samples were collected and mixed at 3, 5, 7 days after the germination. Leaves nuclei were isolated following a previous published protocol with a slight modi cations (Sunaga-Franze et al, 2021;Conde et al, 2021). Approximately 1 g fresh leaf was ground into a powder by freezing with liquid nitrogen.…”