Hemocyte Nuclei Isolation from Adult Drosophila melanogaster for snRNA-seq.

In adult Drosophila, most of the hemocytes are macrophage-like cells (so called plasmatocytes), which serve various functions in organ homeostasis and immune defense. Ontogeny and functions are largely conserved between vertebrate and invertebrate macrophages. Hence, Drosophila offers a powerful genetic toolbox to study macrophage function and genetically modulate these cells. Technological advances in high-throughput sequencing approaches allowed to give an in-depth characterization of vertebrate macrophage populations and their heterogenous composition within different organs as well as changes in disease. Embryonic and larval hemocytes in Drosophila have been recently analyzed in single-cell RNA-sequencing (scRNA-seq) approaches during infection and steady state. These analyses revealed anatomical and functional Drosophila hemocyte subtypes dedicated to specific tasks. Only recently, the Fly Cell Atlas provided a whole transcriptomic single-cell atlas via single-nuclei RNA-sequencing (snRNA-seq) of adult Drosophila including many different tissues and cell types where hemocytes were also included. Yet, a specific protocol to isolate nuclei from adult hemocytes for snRNA-seq and study these cells in different experimental conditions was not available. In this chapter, we give a detailed protocol to purify hemocyte nuclei from adult Drosophila, which can be used in subsequent analyses such as snRNA-seq.

Modified CTAB protocols for high-molecular-weight DNA extractions from ferns.

Premise: Efficient protocols for extracting high-molecular-weight (HMW) DNA from ferns facilitate the long-read sequencing of their large and complex genomes. Here, we perform two cetyltrimethylammonium bromide (CTAB)-based protocols to extract HMW DNA and evaluate their applicability in diverse fern taxa for the first time. Methods and Results: We describe two modified CTAB protocols, with key adjustments to minimize mechanical disruption during lysis to prevent DNA shearing. One of these protocols uses a small amount of fresh tissue but yields a considerable quantity of HMW DNA with high efficiency. The other accommodates a large amount of input tissue, adopts an initial step of nuclei isolation, and thus ensures a high yield in a short period of time. Both methods were proven to be robust and effective in obtaining HMW DNA from diverse fern lineages, including 33 species in 19 families. The DNA extractions mostly had high DNA integrity, with mean sizes larger than 50 kbp, as well as high purity (A260/A230 and A260/A280 > 1.8). Conclusions: This study provides HMW DNA extraction protocols for ferns in the hope of facilitating further attempts to sequence their genomes, which will bridge our genomic understanding of land plant diversity.

The Use of Flow Cytometry for Estimating Genome Sizes and DNA Ploidy Levels in Plants.

Flow cytometry has emerged as a uniquely flexible, accurate, and widely applicable technology for the analysis of plant cells. One of its most important applications centers on the measurement of nuclear DNA contents. This chapter describes the essential features of this measurement, outlining the overall methods and strategies, but going on to provide a wealth of technical details to ensure the most accurate and reproducible results. The chapter is aimed to be equally accessible to experienced plant cytometrists as well as those newly entering the field. Besides providing a step-by-step guide for estimating genome sizes and DNA-ploidy levels from fresh tissues, special attention is paid to the use of seeds and desiccated tissues for such purposes. Methodological aspects regarding field sampling, transport, and storage of plant material are also given in detail. Finally, troubleshooting information for the most common problems that may arise during the application of these methods is provided.

Nuclei on the Rise: When Nuclei-Based Methods Meet Next-Generation Sequencing.

In the last decade, we have witnessed an upsurge in nuclei-based studies, particularly coupled with next-generation sequencing. Such studies aim at understanding the molecular states that exist in heterogeneous cell populations by applying increasingly more affordable sequencing approaches, in addition to optimized methodologies developed to isolate and select nuclei. Although these powerful new methods promise unprecedented insights, it is important to understand and critically consider the associated challenges. Here, we provide a comprehensive overview of the rise of nuclei-based studies and elaborate on their advantages and disadvantages, with a specific focus on their utility for transcriptomic sequencing analyses. Improved designs and appropriate use of the various experimental strategies will result in acquiring biologically accurate and meaningful information.

Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies.

This paper provides a laboratory workflow for single-nucleus RNA-sequencing (snRNA-seq) including a protocol for gentle nuclei isolation from fresh frozen tumor biopsies, making it possible to analyze biobanked material. To develop this protocol, we used non-frozen and frozen human bladder tumors and cell lines. We tested different lysis buffers (IgePal and Nuclei EZ) and incubation times in combination with different approaches for tissue and cell dissection: sectioning, semi-automated dissociation, manual dissociation with pestles, and semi-automated dissociation combined with manual dissociation with pestles. Our results showed that a combination of IgePal lysis buffer, tissue dissection by sectioning, and short incubation time was the best conditions for gentle nuclei isolation applicable for snRNA-seq, and we found limited confounding transcriptomic changes based on the isolation procedure. This protocol makes it possible to analyze biobanked material from patients with well-described clinical and histopathological information and known clinical outcomes with snRNA-seq.

Isolation of Nuclei from Human Snap-frozen Liver Tissue for Single-nucleus RNA Sequencing.

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.

Sample Preparation and Integrative Data Analysis of a Droplet-based Single-Cell ATAC-sequencing Using Murine Thymic Epithelial Cells.

Accessible chromatin regions modulate gene expression by acting as cis-regulatory elements. Understanding the epigenetic landscape by mapping accessible regions of DNA is therefore imperative to decipher mechanisms of gene regulation under specific biological contexts of interest. The assay for transposase-accessible chromatin sequencing (ATAC-seq) has been widely used to detect accessible chromatin and the recent introduction of single-cell technology has increased resolution to the single-cell level. In a recent study, we used droplet-based, single-cell ATAC-seq technology (scATAC-seq) to reveal the epigenetic profile of the transit-amplifying subset of thymic epithelial cells (TECs), which was identified previously using single-cell RNA-sequencing technology (scRNA-seq). This protocol allows the preparation of nuclei from TECs in order to perform droplet-based scATAC-seq and its integrative analysis with scRNA-seq data obtained from the same cell population. Integrative analysis has the advantage of identifying cell types in scATAC-seq data based on cell cluster annotations in scRNA-seq analysis.

Single cell genomics as a transformative approach for aquaculture research and innovation.

Single cell genomics encompasses a suite of rapidly maturing technologies that measure the molecular profiles of individual cells within target samples. These approaches provide a large up-step in biological information compared to long-established 'bulk' methods that profile the average molecular profiles of all cells in a sample, and have led to transformative advances in understanding of cellular biology, particularly in humans and model organisms. The application of single cell genomics is fast expanding to non-model taxa, including aquaculture species, where numerous research applications are underway with many more envisaged. In this review, we highlight the potential transformative applications of single cell genomics in aquaculture research, considering barriers and potential solutions to the broad uptake of these technologies. Focusing on single cell transcriptomics, we outline considerations for experimental design, including the essential requirement to obtain high quality cells/nuclei for sequencing in ectothermic aquatic species. We further outline data analysis and bioinformatics considerations, tailored to studies with the under-characterized genomes of aquaculture species, where our knowledge of cellular heterogeneity and cell marker genes is immature. Overall, this review offers a useful source of knowledge for researchers aiming to apply single cell genomics to address biological challenges faced by the global aquaculture sector though an improved understanding of cell biology.

Multiplexed Single-Nucleus RNA Sequencing Using Lipid-Oligo Barcodes.

This protocol describes a robust pipeline for simultaneously analyzing multiple samples by single-nucleus (sn)RNA-seq. cDNA obtained from each single sample are labeled with the same lipid-coupled oligonucleotide barcode (10X Genomics). Nuclei from as many as 12 individual samples can be pooled together and simultaneously processed for cDNA library construction and subsequent DNA sequencing. While previous protocols using lipid-coupled oligonucleotide barcodes were optimized for analysis of samples consisting of viable cells, this protocol is optimized for analyses of quick-frozen cell samples. The protocol ensures efficient recovery of nuclei both by incorporating high sucrose buffered solutions and by including a tracking dye (trypan blue) during nuclei isolation. The protocol also describes a procedure for removing single nuclei 'artifacts' by removing cell debris prior to single nuclear fractionation. This protocol informs the use of computational tools for filtering poorly labeled nuclei and assigning sample identity using barcode unique molecular identifier (UMI) read counts percentages. The computational pipeline is applicable to either cultured or primary, fresh or frozen cells, regardless of their cell types and species. Overall, this protocol reduces batch effects and experimental costs while enhancing sample comparison. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Labeling cells with lipid oligo barcodes and generating multiplexed single-nucleus RNA-seq libraries Basic Protocol 2: Bioinformatic deconvolution of the multiplexed snRNAseq libraries.

Establishment and Optimization of Molecular Cytogenetic Techniques (45S rDNA-FISH, GISH, and Fiber-FISH) in Kiwifruit (Actinidia Lindl.).

The kiwifruit (Actinidia chinensis) has long been regarded as "the king of fruits" for its nutritional importance. However, the molecular cytogenetics of kiwifruit has long been hampered because of the large number of basic chromosome (x = 29), the inherent small size and highly similar morphology of metaphase chromosomes. Fluorescence in situ hybridization (FISH) is an indispensable molecular cytogenetic technique widely used in many plant species. Herein, the effects of post-hybridization washing temperature on FISH, blocking DNA concentration on genomic in situ hybridization (GISH), extraction method on nuclei isolation and the incubation time on the DNA fiber quality in kiwifruit were evaluated. The post-hybridization washing in 2 × saline sodium citrate (SSC) solution for 3 × 5 min at 37°C ensured high stringency and distinct specific FISH signals in kiwifruit somatic chromosomes. The use of 50 × blocking DNA provided an efficient and reliable means of discriminating between chromosomes derived from in the hybrids of A. chinensis var. chinensis (2n = 2x = 58) × A. eriantha (2n = 2x = 58), and inferring the participation of parental genitors. The chopping method established in the present study were found to be very suitable for preparation of leaf nuclei in kiwifruit. A high-quality linear DNA fiber was achieved by an incubation of 20 min. The physical size of 45S rDNA signals was approximately 0.35-0.40 µm revealed by the highly reproducible fiber-FISH procedures established and optimized in this study. The molecular cytogenetic techniques (45S rDNA-FISH, GISH, and high-resolution fiber-FISH) for kiwifruit was for the first time established and optimized in the present study, which is the foundation for the future genomic and evolutionary studies and provides chromosomal characterization for kiwifruit breeding programs.

High-Resolution ATAC-Seq Analysis of Frozen Clinical Tissues.

The ATAC-seq method enables the genome-wide analysis of accessible chromatin revealing transcriptionally active and poised regulatory elements. The ATAC-seq analysis of clinical specimens at a single-cell resolution reveals the cellular composition of the tissue contributing to the understanding of intra-tissue heterogeneity. Here we describe our method for nuclei isolation from frozen specimens with wide applicability across tissue types, producing nuclei suitable for a number of molecular profiling methods including ATAC-seq in bulk and at a single-cell resolution.

Fractionation and Extraction of Crude Nuclear Proteins From Arabidopsis Seedlings.

The plant nucleus is an important subcellular organelle that contains the genome, ribosomal RNA, and regulatory proteins, and performs a central role in the functioning and metabolism of the cell. Fractionation of intact nuclei is a crucial process to elucidate the function of nuclear proteins. Here, we present a simple method for the fractionation of crude nuclei and extraction of nuclear proteins, based on previously established methods. This protocol provides an easy and quick method to isolate crude nuclei and extract nuclear proteins from Arabidopsis seedlings, which is useful for the research on the nuclear proteins, without requirement for high-purity nuclei. Graphic abstract: Schematic procedure for the isolation of crude nuclei and extraction of nuclear proteins from Arabidopsis seedlings.

Selective isolation of mouse glial nuclei optimized for reliable downstream omics analyses.

BACKGROUND: Isolation of cell types of interest from the brain for molecular applications presents several challenges, including cellular damage during tissue dissociation or enrichment procedures, and low cell number in the tissue in some cases. Techniques have been developed to enrich distinct cell populations using immunopanning or fluorescence activated cell/nuclei sorting. However, these techniques often involve fixation, immunolabeling and DNA staining steps, which could potentially influence downstream omics applications. NEW METHOD: Taking advantage of readily available genetically modified mice with fluorescent-tagged nuclei, we describe a technique for the purification of cell-type specific brain nuclei, optimized to decrease sample preparation time and to limit potential artefacts for downstream omics applications. We demonstrate the applicability of this approach for the purification of glial cell nuclei and show that the resulting cell-type specific nuclei obtained can be used effectively for omics applications, including ATAC-seq and RNA-seq. RESULTS: We demonstrate excellent enrichment of fluorescently-tagged glial nuclei, yielding high quality RNA and chromatin. We identify several critical steps during nuclei isolation that help limit nuclei rupture and clumping, including quick homogenization, dilution before filtration and loosening of the pellet before resuspension, thus improving yield. Sorting of fluorescent nuclei can be achieved without fixation, antibody labelling, or DAPI staining, reducing potential artifactual results in RNA-seq and ATAC-seq analyses. We show that reproducible glial cell type-specific profiles can be obtained in transcriptomic and chromatin accessibility assays using this rapid protocol. COMPARISON WITH EXISTING METHODS: Our method allows for rapid enrichment of glial nuclei populations from the mouse brain with minimal processing steps, while still providing high quality RNA and chromatin required for reliable omics analyses. CONCLUSIONS: We provide a reproducible method to obtain nucleic material from glial cells in the mouse brain with a quick and limited sample preparation.

Isolation of Nuclei from Mammalian Cells and Tissues for Single-Nucleus Molecular Profiling.

Both single-cell RNA sequencing (scRNAseq) and single-nucleus RNA sequencing (snRNAseq) can be used to characterize the transcriptional profile of individual cells, and based on these transcriptional profiles, help define cell type distribution in mixed cell populations. However, scRNAseq analyses are confounded if some of the cells are large (>50 µm) or if some of cells adhere more tightly to some adjacent cells than to others. Further, single cell isolation for scRNAseq requires fresh tissue, which may not be available for human or animal model tissues. Additionally, the current enzymatic and mechanical methods for single-cell dissociation can lead to stress-induced transcriptional artifacts. Nuclei for snRNAseq, on the other hand, can be isolated from any cell, regardless of size, and from either fresh or frozen tissues, and compared to whole cells, they are more resistant to mechanical pressures and appear not to exhibit as many cell isolation-based transcriptional artifacts. Here, we describe a time- and cost-effective procedure to isolate nuclei from mammalian cells and tissues. The protocol incorporates steps to mechanically disrupt samples to release nuclei. Compared to conventional nuclei isolation protocols, the approach described here increases overall efficiency, eliminates risk of contaminant exposure, and reduces volumes of expensive reagents. A series of RNA quality control checks are also incorporated to ensure success and reduce costs of subsequent snRNAseq experiments. Nuclei isolated by this procedure can be separated on the 10× Genomics Chromium system for either snRNAseq and/or Single-Nucleus ATAC-Seq (snATAC-Seq), and is also compatible with other single cell platforms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation and quality control check via RNA Isolation and Analysis Basic Protocol 2: Nuclei Isolation.

Isolation of Plant Nuclei Compatible with Microfluidic Single-nucleus ATAC-sequencing.

Gene expression depends on the binding of transcription factors with DNA regulatory sequences. The level of accessibility for these sequences varies between cells and cell types. Until recently, using the Tn5 assay for transposase-accessible chromatin for sequencing (ATAC-seq) technology allowed assessing the profiles of chromatin from an entire organ or, when coupled with the isolation of nuclei tagged in specific cell types (INTACT) method, from a cell-type. Recently, ATAC-seq experiments were conducted at the level of individual plant nuclei. Applying single nuclei ATAC-seq (sNucATAC-seq) technology to thousands of individual cells revealed more finely tuned profiles of chromatin accessibility. In this manuscript, we describe a method to isolate nuclei fom plant roots and green tissues, permeabilize the nuclear membrane using detergent to allow the penetration of the Tn5 transposase, and re-suspend them in a nuclei resuspension buffer compatible with the construction of sNucATAC-seq libraries using the 10× Genomic's Chromium technology. This protocol was successfully applied on Arabidopsis thaliana and Glycine max root nuclei.

Epigenetic Assays in Purified Cardiomyocyte Nuclei.

The adult mammalian heart's potential for regeneration is very inefficient. Importantly, adult mammalian cardiomyocytes (CMs) are characterized as a cell population with very limited mitotic potential. Conversely, the neonatal mouse heart possesses a brief, yet robust, regenerative capacity within the first week of life. Cell type-specific enrichment procedures are essential for characterizing the full spectrum of epigenomic landscapes and gene regulatory networks deployed by mammalian CMs. In this chapter, we describe a protocol useful for purifying CM nuclei from mammalian cardiac tissue. Furthermore, we detail a low-input procedure suitable for the parallel genome-wide profiling of chromatin accessibility, histone modifications, and transcription factor-binding sites. The CM nuclei purified using this process are suitable for multi-omic profiling approaches.

The Application of Flow Cytometry for Estimating Genome Size, Ploidy Level Endopolyploidy, and Reproductive Modes in Plants.

Over the years, the amount of DNA in a nucleus (genome size) has been estimated using a variety of methods, but increasingly, flow cytometry (FCM) has become the method of choice. The popularity of this technique lies in the ease of sample preparation and in the large number of particles (i.e., nuclei) that can be analyzed in a very short period of time. This chapter presents a step-by-step guide to estimating the nuclear DNA content of plant nuclei using FCM. Attempting to serve as a tool for daily laboratory practice, we list, in detail, the equipment required, specific reagents and buffers needed, as well as the most frequently used protocols to carry out nuclei isolation. In addition, solutions to the most common problems that users may encounter when working with plant material and troubleshooting advice are provided. Finally, information about the correct terminology to use and the importance of obtaining chromosome counts to avoid cytological misinterpretations of the FCM data are discussed.

An optimised chromatin immunoprecipitation (ChIP) method for starchy leaves of Nicotiana benthamiana to study histone modifications of an allotetraploid plant.

All flowering plants have evolved through multiple rounds of polyploidy throughout the evolutionary process. Intergenomic interactions between subgenomes in polyploid plants are predicted to induce chromatin modifications such as histone modifications to regulate expression of gene homoeologs. Nicotiana benthamiana is an ancient allotetraploid plant with ecotypes collected from climatically diverse regions of Australia. Studying the chromatin landscape of this unique collection will likely shed light on the importance of chromatin modifications in gene regulation in polyploids as well its implications in adaptation of plants in environmentally diverse conditions. Generally, chromatin immunoprecipitation and high throughput DNA sequencing (ChIP-seq) is used to study chromatin modifications. However, due to the starchy nature of mature N. benthamiana leaves, previously published protocols were unsuitable. The higher amounts of starch in leaves that co-precipitated with nuclei hindered downstream processing of DNA. Here we present an optimised ChIP protocol for N. benthamiana leaves to facilitate comparison of chromatin modifications in two closely related ecotypes. Several steps of ChIP were optimised including tissue harvesting, nuclei isolation, nuclei storage, DNA shearing and DNA recovery. Commonly available antibodies targeting histone 3 lysine 4 trimethylation (H3K4me3) and histone 3 lysine 9 dimethylation (H3K9me2) histone modifications were used and success of ChIP was confirmed by PCR and next generation sequencing. Collectively, our optimised method is the first comprehensive ChIP method for mature starchy leaves of N. benthamiana to enable studies of chromatin landscape at the genome-wide scale.

Quick and facile preparation of histone proteins from the green microalga Chlamydomonas reinhardtii and other photosynthetic organisms.

The development of universal, broadly applicable methods for histone extraction from animal cells and tissues has unlocked the ability to compare these epigenetic-influencing proteins across tissue types, healthy and diseased states, and cancerous versus normal cells. However, for plants and green algae, a quick and easily implemented histone extraction method has yet to be developed. Here, we report an optimized method that provides a unified approach to extract histones for the green microalgal species Chlamydomonas reinhardtii and Scenedesmus dimorphus as well as for maize (corn) leaf tissue. Histone extraction methods include treatment with high salt concentrations and acidification. Preparations of nuclei can be made in ∼3.5 h and histones extracted in ∼3.5 h either immediately or nuclei may be frozen and histone proteins can be later extracted without a change in histone PTM patterns. To examine the efficiency of the new methods provided, we performed both qualitative and quantitative analysis of salt and acid-extracted whole histone proteins (SAEWH) via SDS-PAGE gel electrophoresis and intact protein mass spectrometry. SDS-PAGE analysis indicated that histone yields decrease when using walled Chlamydomonas strains relative to cell-wall-less mutants. Using top-down mass spectrometry (TDMS) for intact protein analysis, we confirmed the presence of H4K79me1 in multiple algal species; however, this unique modification was not identified in corn leaf tissue and has not been reported elsewhere. TDMS measurements of SAEWH extracts also revealed that oxidation which occurs during the histone extraction process does not increase with exposure of harvested algal cells, their nuclei and the extracted histone samples to light.

Generation of an equine biobank to be used for Functional Annotation of Animal Genomes project.

The Functional Annotation of Animal Genomes (FAANG) project aims to identify genomic regulatory elements in both sexes across multiple stages of development in domesticated animals. This study represents the first stage of the FAANG project for the horse, Equus caballus. A biobank of 80 tissue samples, two cell lines and six body fluids was created from two adult Thoroughbred mares. Ante-mortem assessments included full physical examinations, lameness, ophthalmologic and neurologic evaluations. Complete blood counts and serum biochemistries were also performed. At necropsy, in addition to tissue samples, aliquots of serum, ethylenediaminetetraacetic acid (EDTA) plasma, heparinized plasma, cerebrospinal fluid, synovial fluid, urine and microbiome samples from all regions of the gastrointestinal and urogenital tracts were collected. Epidermal keratinocytes and dermal fibroblasts were cultured from skin samples. All tissues were grossly and histologically evaluated by a board-certified veterinary pathologist. The results of the clinical and pathological evaluations identified subclinical eosinophilic and lymphocytic infiltration throughout the length of the gastrointestinal tract as well as a mild clinical lameness in both animals. Each sample was cryo-preserved in multiple ways, and nuclei were extracted from selected tissues. These samples represent the first published systemically healthy equine-specific biobank with extensive clinical phenotyping ante- and post-mortem. The tissues in the biobank are intended for community-wide use in the functional annotation of the equine genome. The use of the biobank will improve the quality of the reference annotation and allow all equine researchers to elucidate unknown genomic and epigenomic causes of disease.