FlsnRNA-seq: protoplasting-free full-length single-nucleus RNA profiling in plants

Long, Yanping; Liu, Zhijian; Jia, Jinbu; Mo, Weipeng; Fang, Liang; Lu, Dongdong; Liu, Bo; Hong, Zhang; Chen, Wei; Zhai, Jixian

doi:10.1186/s13059-021-02288-0

Cited by 74 publications

(54 citation statements)

References 71 publications

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“…Although cell walls can be removed by enzymatic treatment of tissues that are easy to access, such protoplasting techniques remain impractical for early embryos because they are deeply embedded within maternal seed tissues. Single-nucleus mRNA-sequencing (snRNA-seq) ( Habib et al, 2016 ) offers an alternative method to inspect transcriptomes at single-cell resolution in plants and has been recently applied to roots ( Farmer et al, 2021 ) and endosperm tissues within seeds ( Long et al, 2021 ; Picard et al, 2021 ). Here, we present a workflow to obtain contamination-free high-quality transcriptomes from individual early embryonic nuclei followed by their assignments to the precursors of the most fundamental plant tissues including the shoot meristem, distal regions of the root meristem and epidermal and vascular tissues.…”

Section: Introductionmentioning

confidence: 99%

Gene expression variation in Arabidopsis embryos at single-nucleus resolution

et al. 2021

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Soon after fertilization of egg and sperm, plant genomes become transcriptionally activated and drive a series of coordinated cell divisions to form the basic body plan during embryogenesis. Early embryonic cells rapidly diversify from each other, and investigation of the corresponding gene expression dynamics can help elucidate underlying cellular differentiation programs. However, current plant embryonic transcriptome datasets either lack cell-specific information or have RNA contamination from surrounding non-embryonic tissues. We have coupled fluorescence-activated nuclei sorting together with single-nucleus mRNA sequencing to construct a gene expression atlas of Arabidopsis thaliana early embryos at single-cell resolution. In addition to characterizing cell-specific transcriptomes, we found evidence that distinct epigenetic and transcriptional regulatory mechanisms operate across emerging embryonic cell types. These datasets and analyses, as well as the approach we devised, are expected to facilitate the discovery of molecular mechanisms underlying pattern formation in plant embryos.

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Section: Introductionmentioning

confidence: 99%

Gene expression variation in Arabidopsis embryos at single-nucleus resolution

et al. 2021

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“…The ratio was 1.3 for pluripotent stem cells [ 24 ], 1.49 for human brain nuclei, and 1.66 for mouse brain nuclei [ 26 ]. Very recently, a ratio of 1.40 was reported for a nuclei isolation protocol using Arabidopsis roots [ 27 ]. We found a ratio of 1.46, 1.63, and 1.62 for SAM, and stem replicates 1 and 2, respectively.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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Single-cell transcriptome analysis has been extensively applied in humans and animal models to uncover gene expression heterogeneity between the different cell types of a tissue or an organ. It demonstrated its capability to discover key regulatory elements that determine cell fate during developmental programs. Single-cell analysis requires the isolation and labeling of the messenger RNA (mRNA) derived from each cell. These challenges were primarily addressed in mammals by developing microfluidic-based approaches. For plant species whose cells contain cell walls, these approaches have generally required the generation of isolated protoplasts. Many plant tissues’ secondary 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. Consequently, single-cell RNA sequencing (scRNA-seq) studies using microfluidic approaches in plants have mainly been restricted to Arabidopsis roots, for which well-established procedures of protoplast isolation are available. Here we present a simple alternative approach to generating high-quality protoplasts from plant tissue by characterizing the mRNA extracted from individual nuclei instead of whole cells. We developed the protocol using two different plant materials with varying cellular complexity levels and cell wall structure, Populus shoot apices, and more lignified stems. Using the 10× Genomics Chromium technology, we show that this procedure results in intact mRNA isolation and limited leakage, with a broad representation of individual cell transcriptomes.

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“…It increases their resolution from the small cell population to the single cell level. Successful combinations of FACS [195], FANS [196,197] and INTACT [198] with commercial droplet-based platform were reported (see Table S1). Therefore, there is no doubt that studies combining the TRAP approach with 10X Genomics barcode technology may appear soon.…”

Section: Single-cell Transcriptomicsmentioning

confidence: 99%

OMICs, Epigenetics, and Genome Editing Techniques for Food and Nutritional Security

Gogolev

Ahmar

Akpınar³

et al. 2021

Plants

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The incredible success of crop breeding and agricultural innovation in the last century greatly contributed to the Green Revolution, which significantly increased yields and ensures food security, despite the population explosion. However, new challenges such as rapid climate change, deteriorating soil, and the accumulation of pollutants require much faster responses and more effective solutions that cannot be achieved through traditional breeding. Further prospects for increasing the efficiency of agriculture are undoubtedly associated with the inclusion in the breeding strategy of new knowledge obtained using high-throughput technologies and new tools in the future to ensure the design of new plant genomes and predict the desired phenotype. This article provides an overview of the current state of research in these areas, as well as the study of soil and plant microbiomes, and the prospective use of their potential in a new field of microbiome engineering. In terms of genomic and phenomic predictions, we also propose an integrated approach that combines high-density genotyping and high-throughput phenotyping techniques, which can improve the prediction accuracy of quantitative traits in crop species.

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FlsnRNA-seq: protoplasting-free full-length single-nucleus RNA profiling in plants

Cited by 74 publications

References 71 publications

Gene expression variation in Arabidopsis embryos at single-nucleus resolution

Gene expression variation in Arabidopsis embryos at single-nucleus resolution

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

OMICs, Epigenetics, and Genome Editing Techniques for Food and Nutritional Security

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