Cross-species optimization of nuclei isolation in ten plant species.

Chapter 4 Isolation of Nuclei and Preparation of Chromatin from Plant Tissues

e17535 Background: High-grade serous ovarian carcinoma (HGSOC) is the most frequent and deadliest type of ovarian cancer (OC). With the recent advances in transcriptomic and epigenomic profiling of cancer at single cell resolution, it has become clear that intra-tumor heterogeneity driven by genetic and epigenetic factors may be the basis of drug resistance. Current treatments are not homogeneously effective against all the cancer cell subpopulations, thus enabling resistance. With single cell sequencing technology, characterization of cell signatures and regulatory mechanisms may translate to prognostic markers and novel drug targets. Methods: The transcriptomic and epigenomic landscape of HGSOC was mapped for five tumor samples from debulking surgeries and one tissue patient-derived xenograft (PDX) using the 10X genomics sequencing platform. This strategy permits the combination of single cell ATAC-seq and single cell RNA-seq within the same nucleus (Multiome). Isolation of nuclei, library preparation and sequencing were performed following 10X genomics protocols. Sample quality and library quality were assessed by nuclear morphology and fragment size analysis, respectively. Sequenced reads were mapped to the human genome and downstream analysis including copy number variant prediction, pathway enrichment, motif enrichment and gene regulatory network analysis were performed. The sampled patients continue to be followed for clinical outcomes, such as response to therapy. Clinical data including patient age, grade, and stage of cancer, debulking status, CA-125 levels, and neoadjuvant status, were used in conjunction with the genomics data to characterize patient specific molecular regulatory signatures. Results: Cells from the six samples (total N= 26,421) were projected via UMAP. Immune and stromal cells were shown to cluster by cell-type while cancer cells clustered by patient. Cancer cells were identified as cycling cells, ciliated cells, cells with enrichment of the JAK-STAT signaling pathway, and cells exhibiting cancer stemness signatures. Transcription factor motifs and binding-site enrichment in open chromatin regions reveal transcription regulation-based subpopulations. Using the combined gene expression and open chromatin information from these cells, there is potential to uncover the genetic regulatory network(s) that drives treatment resistance. Conclusions: With single cell technology, specific clusters of cancer and tumor microenvironment cells can be classified. Beyond characterizing patient specific signatures, multiome enables the discovery of genomic, transcriptomic and epigenomic signatures that provide insight in tumor progression and treatment resistance.

Single-nucleus RNA sequencing (snRNA-seq) provides a powerful tool for studying cell type composition in heterogenous tissues. The liver is a vital organ composed of a diverse set of cell types; thus, single-cell technologies could greatly facilitate the deconvolution of liver tissue composition and various downstream omics analyses at the cell-type level. Applying single-cell technologies to fresh liver biopsies can, however, be very challenging, and snRNA-seq of snap-frozen liver biopsies requires some optimization given the high nucleic acid content of the solid liver tissue. Therefore, an optimized protocol for snRNA-seq specifically targeted for the use of frozen liver samples is needed to improve our understanding of human liver gene expression at the cell-type resolution. We present a protocol for performing nuclei isolation from snap-frozen liver tissues, as well as guidance on the application of snRNA-seq. We also provide guidance on optimizing the protocol to different tissue and sample types.

Estimation of the <i>Glomus intraradices</i> nuclear DNA content

Single cell technologies have advanced at a rapid pace, providing assays for various molecular phenotypes. Droplet-based single cell technologies, particularly those based on nuclei isolation, such as simultaneous RNA+ATAC single-cell multiome, are susceptible to exogenous ambient molecule contamination, which can increase noise in cell type-level associations. We reasoned that genotype-based sample multiplexing can provide an opportunity to infer this ambient contamination by leveraging DNA variation in sequenced reads. Thus, we developed ambimux, a likelihood-based method to estimate ambient fractions and demultiplex single-cell multiome experiments using genotype-level data. Ambimux models the ambient or nuclear probability at the read level and thus can classify empty droplets and estimate droplet-specific ambient molecule fractions in each modality. We first evaluated our method using simulated data sets across a range of parameters. We found that ambimux closely estimated the ground truth droplet contamination fractions in the RNA (MAE=0.048) and ATAC (MAE=0.042) modalities. As a result, ambimux maintained high specificity (>95%) and was able to correctly assign singlets at considerably high ambient fractions (up to 60%) for both RNA and ATAC modalities. In comparison with models that do not consider ambient contamination, these only maintained similar sensitivity levels at considerably lower ambient fractions (up to 25%). We then generated a real data set of seven visceral adipose tissue biopsies run on a single 10x Multiome channel. We ran ambimux and detected 4,986 singlets, capturing similar numbers as other methods. Then, we sought to evaluate the fidelity of the ambient fraction estimates from ambimux. We split singlets into ambient-enriched (>5% contamination in both modalities) or nuclear-enriched (<5% in both) droplets and performed gene-peak linkage analysis. Low ambient droplets resulted in more significant hits with gene-peak links enriched at the transcription start site relative to high ambient droplets, suggesting that the ambient droplets identified by ambimux hamper the identification of biologically meaningful signals. In summary, we developed a joint single-cell multiome demultiplexing method, ambimux, that accurately models and estimates ambient molecule contamination in each modality.

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 nanowell-based 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.

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy homeostasis and the metabolism of xenobiotics, among others. These tasks are performed through tight coordination between hepatic parenchymal and non-parenchymal cells. Additionally, various metabolic activities are confined to specific areas of the hepatic lobule-a phenomenon called liver zonation. Recent advances in single-cell sequencing technologies have empowered researchers to investigate tissue heterogeneity at a single-cell resolution. In many complex tissues, including the liver, harsh enzymatic and/or mechanical dissociation protocols can negatively affect the viability or the quality of the single-cell suspensions needed to comprehensively characterize this organ in health and disease. This paper describes a robust and reproducible protocol for isolating nuclei from frozen, archived liver tissues. This method yields high-quality nuclei that are compatible with downstream, single-cell omics approaches, including single-nucleus RNA-seq, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), as well as multimodal omics (joint RNA-seq and ATAC-seq). This method has been successfully used for the isolation of nuclei from healthy and diseased human, mouse, and non-human primate frozen liver samples. This approach allows the unbiased isolation of all the major cell types in the liver and, therefore, offers a robust methodology for studying the liver at the single-cell resolution.

Evaluating the relationship between floristic quality and measures of plant biodiversity along stream bank habitats

Savannas with scattered oak canopies, once the most widespread communities in southern Wisconsin, now are among the most endangered. Surprisingly little is known about the composition, structure, and horizontal patterning of their species-rich ground layers. This study relates the distribution and ecological characteristics of 417 ground-layer species to local and regional gradients in soil composition and light regime, based on an analysis of 722 1-m2 quadrats in 12 remnant savannas. Our findings have important implications for efforts to restore/conserve midwestern oak savannas. Ground-layer composition was strongly related to among-site differences in soil texture and within-site differences in light availability, with variation in sand vs. silt content accounting for twice as much turnover in species composition as that accounted for by direct photon flux density (PFD) estimated from hemispherical photographs. Most species reached peak coverage under sunny or partly shaded conditions. Flowering/fruiting was often skewed toward sunnier microsites. Absolute forb cover increased with silt content and declined with PFD. Graminoid cover showed a curvilinear relationship to soil texture and light, being highest in well-lit, moderately sandy microsites. Total ground-layer cover increased with silt content at a given irradiance; it increased with PFD on silty sites and decreased with PFD on sandy sites. Forb cover increased regularly with PFD and sand content. When quadrats were stratified by sand content and PFD, species richness of forbs and graminoids increased linearly with coverage by each group, with far more forb species present at a given coverage. Among graminoids, C4 grasses were common only in bright, sandy microsites; C3 grasses and sedges had broader ecological distributions. Among forbs, leaf width increased and leaf inclination became more horizontal toward shadier and siltier microsites; tall herbs were generally found in silty areas with dense ground-layer cover. Plants with N-fixing symbioses were found mostly on sandy, well-lit microsites, although climbing species occured on shadier and/or siltier microsites, where N-fixation might be expected to be less advantageous. Most ground-layer species were perennial (88%), with few annuals (6%) or biennials (6%). For the 85 most common species, breadth of distribution across savanna microhabitats (4 soil × 4 light) was significantly correlated with presence across 34 Wisconsin community types, suggesting that similar factors help to constrain distributions at local and regional scales. Species in the two largest genera (Aster and Solidago) differed significantly in distribution according to the Syrjala test, supporting habitat partitioning as a mechanism of coexistence. Oak savannas are unusually diverse. At small spatial scales, there were 16.1 ± 1.3 species/m2 (mean ± 1 sd), compared with 11.4 ± 2.7 for prairies and 8.2 ± 2.5 for forests. At slightly larger scales, savannas showed high spatial turnover in ground-layer composition: 89.3 ± 12.0 species/20 m2, compared with 41.5 ± 8.3 for prairies and 42.8 ± 8.0 for forests. At large spatial scales, a survey of 22 savanna remnants (42 ha) showed extensive floristic differentiation: 507 native plant species, ∼27% of Wisconsin's indigenous vascular flora (∼14 × 106 ha). Contrary to previous reports, Midwest oak savannas are forb dominated, except on the sandiest or sunniest microsites. Release of forbs from competition with C4 grasses under partly shaded conditions may help to account for the high diversity of savanna ground layers relative to prairies. Divergent distributions of plants with different adaptations for energy capture, together with large variation within sites in ground-layer light regime and among sites in soil texture, suggest that partitioning of light and soil gradients is important for maintaining the high plant diversity of oak savannas. Mass effects (involving dispersal subsidies from favorable microsites) may also play a role in maintaining diversity within these mosaic communities.

Savannas with scattered oak canopies, once the most widespread communities in southern Wisconsin, now are among the most endangered. Surprisingly little is known about the composition, structure, and horizontal patterning of their species-rich ground layers. This study relates the distribution and ecological characteristics of 417 ground-layer species to local and regional gradients in soil composition and light regime, based on an analysis of 722 1-m2 quadrats in 12 remnant savannas. Our findings have important implications for efforts to restore/conserve midwestern oak savannas. Ground-layer composition was strongly related to among-site differences in soil texture and within-site differences in light availability, with variation in sand vs. silt content accounting for twice as much turnover in species composition as that accounted for by direct photon flux density (PFD) estimated from hemispherical photographs. Most species reached peak coverage under sunny or partly shaded conditions. Flowering/fruiting was often skewed toward sunnier microsites. Absolute forb cover increased with silt content and declined with PFD. Graminoid cover showed a curvilinear relationship to soil texture and light, being highest in well-lit, moderately sandy microsites. Total ground-layer cover increased with silt content at a given irradiance; it increased with PFD on silty sites and decreased with PFD on sandy sites. Forb cover increased regularly with PFD and sand content. When quadrats were stratified by sand content and PFD, species richness of forbs and graminoids increased linearly with coverage by each group, with far more forb species present at a given coverage. Among graminoids, C4 grasses were common only in bright, sandy microsites; C3 grasses and sedges had broader ecological distributions. Among forbs, leaf width increased and leaf inclination became more horizontal toward shadier and siltier microsites; tall herbs were generally found in silty areas with dense ground-layer cover. Plants with N-fixing symbioses were found mostly on sandy, well-lit microsites, although climbing species occured on shadier and/or siltier microsites, where N-fixation might be expected to be less advantageous. Most ground-layer species were perennial (88%), with few annuals (6%) or biennials (6%). For the 85 most common species, breadth of distribution across savanna microhabitats (4 soil × 4 light) was significantly correlated with presence across 34 Wisconsin community types, suggesting that similar factors help to constrain distributions at local and regional scales. Species in the two largest genera (Aster and Solidago) differed significantly in distribution according to the Syrjala test, supporting habitat partitioning as a mechanism of coexistence. Oak savannas are unusually diverse. At small spatial scales, there were 16.1 ± 1.3 species/m2 (mean ± 1 sd), compared with 11.4 ± 2.7 for prairies and 8.2 ± 2.5 for forests. At slightly larger scales, savannas showed high spatial turnover in ground-layer composition: 89.3 ± 12.0 species/20 m2, compared with 41.5 ± 8.3 for prairies and 42.8 ± 8.0 for forests. At large spatial scales, a survey of 22 savanna remnants (42 ha) showed extensive floristic differentiation: 507 native plant species, ∼27% of Wisconsin's indigenous vascular flora (∼14 × 106 ha). Contrary to previous reports, Midwest oak savannas are forb dominated, except on the sandiest or sunniest microsites. Release of forbs from competition with C4 grasses under partly shaded conditions may help to account for the high diversity of savanna ground layers relative to prairies. Divergent distributions of plants with different adaptations for energy capture, together with large variation within sites in ground-layer light regime and among sites in soil texture, suggest that partitioning of light and soil gradients is important for maintaining the high plant diversity of oak savannas. Mass effects (involving dispersal subsidies from favorable microsites) may also play a role in maintaining diversity within these mosaic communities.

Single-cell multiomics technologies have significantly advanced our understanding of cellular heterogeneity and biological complexity. The joint profiling of single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq) within the same cell offers a powerful approach for enabling direct linkage between gene expression and chromatin accessibility at single-cell resolution. This integration significantly enhances sensitivity and specificity in identifying rare cell populations and elucidating epigenetic regulatory mechanisms. In this study, we present a robust, high-throughput droplet-based microfluidic protocol that enables simultaneous profiling of RNA and chromatin accessibility from individual cells. The streamlined workflow incorporates key steps, including cell pretreatment, nuclei isolation, gel bead-in-emulsion (GEM) generation, and the construction of scRNA-seq and scATAC-seq libraries. The protocol supports parallel processing of tens of thousands of cells in a single experiment, offering exceptional scalability and reproducibility. The multimodal nature of this approach allows for integrative analysis of multiple features from the same cell, making it an invaluable tool for dissecting complex biological systems.

Studying brain aging at single-cell resolution in vertebrate systems remains challenging due to cost, time, and technical constraints. Here, we demonstrate a protocol to generate single-nucleus RNA sequencing (snRNA-seq) libraries from the brains of the naturally short-lived vertebrate African turquoise killifish Nothobranchius furzeri. The African turquoise killifish has a lifespan of 4-6 months and can be housed in a cost-effective manner, thus reducing cost and time barriers to study vertebrate brain aging. However, tailored protocols are needed to isolate nuclei of sufficient quality for downstream single-cell experiments from the brain of young and aged fish. Here, we demonstrate an empirically optimized protocol for the isolation of high-quality nuclei from the brain of adult African turquoise killifish, a critical step in the generation of high-quality single nuclei omic libraries. Furthermore, we show that the steps to reduce contaminating background RNA are important to clearly distinguish cell types. In summary, this protocol demonstrates the feasibility of studying brain aging in non-traditional vertebrate model organisms.

Studying brain aging at single-cell resolution in vertebrate systems remains challenging due to cost, time, and technical constraints. Here, we demonstrate a protocol to generate single-nucleus RNA sequencing (snRNA-seq) libraries from the brains of the naturally short-lived vertebrate African turquoise killifish Nothobranchius furzeri. The African turquoise killifish has a lifespan of 4-6 months and can be housed in a cost-effective manner, thus reducing cost and time barriers to study vertebrate brain aging. However, tailored protocols are needed to isolate nuclei of sufficient quality for downstream single-cell experiments from the brain of young and aged fish. Here, we demonstrate an empirically optimized protocol for the isolation of high-quality nuclei from the brain of adult African turquoise killifish, a critical step in the generation of high-quality single nuclei omic libraries. Furthermore, we show that the steps to reduce contaminating background RNA are important to clearly distinguish cell types. In summary, this protocol demonstrates the feasibility of studying brain aging in non-traditional vertebrate model organisms.

Abiotic and biotic factors explain independent gradients of plant community composition in ponderosa pine forests

Pollinators play a key role in the reproduction of most plant species, and pollinator and plant diversity are often related. We studied an experimental gradient of plant species richness for a better understanding of plant–pollinator community interactions and their temporal variability, because in non‐experimental field surveys plant richness is often confounded with gradients in management, soil fertility, and community composition. We observed pollinator species richness and frequency of visits six times in 73 plots over two years, and used advanced statistical analysis to account for the high number of zeroes that often occur in count data of rare species. The frequency of pollinator visits increased linearly with both the blossom cover and the number of flowering plant species, which was closely related to the total number of plant species, whereas the number of pollinator species followed a saturation curve. The presence of particularly attractive plant species was only important for the frequency of flower visits, but not to the richness of pollinators. Plant species richness, blossom cover, and the presence of attractive plant species enhanced the temporal stability in the frequency of pollinator visits. In conclusion, grasslands with high plant diversity enhance and stabilize frequent and diverse flower visitations, which should sustain effective pollination and plant reproduction.