A robust method of nuclei isolation for single-cell RNA sequencing of solid tissues from the plant genus Populus

We present a simple alternative approach to generating high-quality protoplasts from plant tissue by characterizing the messenger RNA extracted from individual nuclei instead of whole cells

Daniel Conde; Paolo M. Triozzi; Kelly M. Balmant; Andria L. Doty; Mariza Miranda; Anthony Boullosa; Henry W. Schmidt; Wendell J. Pereira; Christopher Dervinis; Matias Kirst


Scholarcy highlights

  • We describe the optimizations made to this protocol for posterior sequencing of RNA extracted from nuclei. These optimizations had three primary goals: minimize nuclei membrane damage to reduce messenger RNA leakage; avoid mRNA degradation that results in a low-quality cDNA; limit organelles’ contamination, such as chloroplast, and remove cellular debris that interferes with microfluidic devices used in posterior sample preparation
  • We aimed to develop a nuclei isolation protocol for RNA sequencing, that overcomes the difficulties of generating protoplasts from solid plant tissues
  • Plant tissuessecondary cell wall hinders enzymatic digestion required for individual protoplast isolation, resulting in an unequal representation of cell types in a protoplast population. This limitation is especially critical for cell types located in the inner layers of a tissue or the inner tissues of an organ
  • Alternative approaches for transcriptome characterization of individual cells that bypass the need for enzymatic digestion and protoplast isolation are necessary
  • We tested three different nuclei isolation methods for the plant material described in this work, which resulted in suboptimal nuclei integrity preservation, mRNA leaking, or high buffer stickiness during the Fluorescence Activated Nuclei Sorting step
  • To verify our nuclei isolation protocol’s suitability for its application in single-cell transcriptome analysis, we prepared three snRNA-seq libraries using the 10× Genomics Chromium technology

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