The kidney glomerulus is essential for proper kidney function. Until recently, technical challenges associated with glomerular isolation and subsequent dissolution into single cells have limited the detailed characterization of cells in the glomerulus. Previous techniques of kidney dissociation result in low glomerular cell yield, which limits high-throughput analysis. The ability to efficiently purify

Profiling chromatin accessibility at the single-cell level provides critical information about cell type composition and cell-to-cell variation within a complex tissue. Emerging techniques for the interrogation of chromatin accessibility in individual cells allow investigation of the fundamental mechanisms that lead to the variability of different cells. This protocol describes a fast and robust method

Mass-spectrometry-based proteomic analysis is a powerful approach for discovering new disease biomarkers. However, certain critical steps of study design such as cohort selection, evaluation of statistical power, sample blinding and randomization, and sample/data quality control are often neglected or underappreciated during experimental design and execution. This tutorial discusses important steps

The integration of DNA methylation and transcriptional state within single cells is of broad interest. Several single-cell dual- and multi-omics approaches have been reported that enable further investigation into cellular heterogeneity, including the discovery and in-depth study of rare cell populations. Such analyses will continue to provide important mechanistic insights into the regulatory consequences

Single-cell RNA-sequencing data have significantly advanced the characterization of cell-type diversity and composition. However, cell-type definitions vary across data and analysis pipelines, raising concerns about cell-type validity and generalizability. With MetaNeighbor, we proposed an efficient and robust quantification of cell-type replicability that preserves dataset independence and is highly

Naive human pluripotent stem cells (hPSCs) can be used to generate mature human cells of all three germ layers in mouse–human chimeric embryos. Here, we describe a protocol for generating mouse–human chimeric embryos by injecting naive hPSCs converted from the primed state. Primed hPSCs are treated with a mammalian target of rapamycin inhibitor (Torin1) for 3 h and dissociated to single cells, which

The minimal inhibitory concentration (MIC) assay uses agar or broth dilution methods to measure, under defined test conditions, the lowest effective concentration of an antimicrobial agent that inhibits visible growth of a bacterium of interest. This assay is used to test the susceptibilities of bacterial isolates and of novel antimicrobial drugs, and is typically done in nutrient-rich laboratory media

Thrombin generation (TG) assays are used widely to investigate both diseases and drugs that impact thrombosis and bleeding. TG assays were also instrumental in the identification of thrombogenic impurities in immune globulin products, which were associated with thrombotic adverse events in patients. TG assays are therefore now used by quality control laboratories of plasma derivative drug manufacturers

Advances in multiplexed imaging technologies have drastically improved our ability to characterize healthy and diseased tissues at the single-cell level. Co-detection by indexing (CODEX) relies on DNA-conjugated antibodies and the cyclic addition and removal of complementary fluorescently labeled DNA probes and has been used so far to simultaneously visualize up to 60 markers in situ. CODEX enables

Many supramolecular materials in biological systems are driven to a nonequilibrium state by the irreversible consumption of high-energy molecules such as ATP or GTP. As a result, they exhibit unique dynamic properties such as a tunable lifetime, adaptivity or the ability to self-heal. In contrast, synthetic counterparts that exist in or close to equilibrium are controlled by thermodynamic parameters

The presence of microbes in the colon impacts host physiology. Therefore, microbes are being evaluated as potential treatments for colorectal diseases. Humanized model systems that enable robust culture of primary human intestinal cells with bacteria facilitate evaluation of potential treatments. Here, we describe a protocol that can be used to coculture a primary human colon monolayer with aerotolerant

Advances in single-cell genomics now enable large-scale comparisons of cell states across two or more experimental conditions. Numerous statistical tools are available to identify individual genes, proteins or chromatin regions that differ between conditions, but many experiments require inferences at the level of cell types, as opposed to individual analytes. We developed Augur to prioritize the cell

Deaminase fused-Cas9 base editing technologies have enabled precise single-nucleotide genomic editing without the need for the introduction of damaging double-stranded breaks and inefficient homology-directed repair. However, current methods to isolate base-edited cell populations are ineffective, especially when utilized with human pluripotent stem cells, a cell type resistant to genome modification

The dimensionality of cytometry data has strongly increased in the last decade, and in many situations the traditional manual downstream analysis becomes insufficient. The field is therefore slowly moving toward more automated approaches, and in this paper we describe the protocol for analyzing high-dimensional cytometry data using FlowSOM, a clustering and visualization algorithm based on a self-organizing

The dynamics of DNA double-strand break (DSB) repairs including homology-directed repair and nonhomologous end joining play an important role in diseases and therapies. However, investigating DSB repair is typically a low-throughput and cross-sectional process, requiring disruption of cells and organisms for subsequent nuclease-, sequencing- or reporter-based assays. In this protocol, we provide instructions

Deep learning offers the potential to extract more than meets the eye from images captured by imaging flow cytometry. This protocol describes the application of deep learning to single-cell images to perform supervised cell classification and weakly supervised learning, using example data from an experiment exploring red blood cell morphology. We describe how to acquire and transform suitable input

Extracellular vesicles (EVs) are heterogeneous lipid containers with a complex molecular cargo comprising several populations with unique roles in biological processes. These vesicles are closely associated with specific physiological features, which makes them invaluable in the detection and monitoring of various diseases. EVs play a key role in pathophysiological processes by actively triggering

Radiolabeled antibodies have shown promise as tools for both the nuclear imaging and endoradiotherapy of cancer, but the protracted circulation time of radioimmunoconjugates can lead to high radiation doses to healthy tissues. To circumvent this issue, we have developed an approach to positron emission tomography (PET) imaging and radioimmunotherapy (RIT) predicated on radiolabeling the antibody after

The l-tryptophan–derived tricyclic hydroperoxide cis-WOOH was recently identified as a novel and biologically important factor for regulating vascular tone and blood pressure under inflammatory conditions and potentially other cellular redox signaling events. cis-WOOH is highly labile and currently not available commercially. In this protocol, we provide procedures for the synthesis, purification,

Raman spectroscopy can provide a rapid, label-free, nondestructive measurement of the chemical fingerprint of a sample and has shown potential for cancer screening and diagnosis. Here we report a protocol for Raman microspectroscopic analysis of different exfoliative cytology samples (cervical, oral and lung), covering sample preparation, spectral acquisition, preprocessing and data analysis. The protocol

More than 90% of the human genome is transcribed into noncoding RNAs, but their functional characterization has lagged behind. A major bottleneck in the understanding of their functions and mechanisms has been a dearth of systematic methods for identifying interacting protein partners. There now exist several methods, including identification of direct RNA interacting proteins (iDRiP), chromatin isolation

How dynamic activity in neural circuits gives rise to behavior is a major area of interest in neuroscience. A key experimental approach for addressing this question involves measuring extracellular neuronal activity in awake, behaving animals. Recently developed Neuropixels probes have provided a step change in recording neural activity in large tissue volumes with high spatiotemporal resolution. This

Super-resolution fluorescence imaging that surpasses the classical optical resolution limit is widely utilized for resolving the spatial organization of biological structures at molecular length scales. In one example, single-molecule localization microscopy, the lateral positions of single molecules can be determined more precisely than the diffraction limit if the camera collects individual photons

Glycosphingolipids (GSLs) are ubiquitous glycoconjugates present on the cell membrane; they play significant roles in many bioprocesses such as cell adhesion, embryonic development, signal transduction and carcinogenesis. Analyzing such amphiphilic molecules is a major challenge in the field of glycosphingolipidomics. We provide a step-by-step protocol that uses a lectin microarray to analyze GSL glycans

Most brain functions engage a network of distributed regions. Full investigation of these functions thus requires assessment of whole brains; however, whole-brain functional imaging of behaving animals remains challenging. This protocol describes how to follow brain-wide activity in awake head-fixed mice using functional ultrasound imaging, a method that tracks cerebral blood volume dynamics. We describe

The most common nonstandard nucleotides found in genomic DNA are ribonucleotides. Although ribonucleotides are frequently incorporated into DNA by replicative DNA polymerases, very little is known about the distribution and signatures of ribonucleotides incorporated into DNA. Recent advances in high-throughput ribonucleotide sequencing can capture the exact locations of ribonucleotides in genomic DNA

Knowledge of the effect of foods on gut microbiota composition and functionality is expanding. To isolate the effect of single foods and/or single nutrients (i.e., fiber, polyphenols), this protocol describes an in vitro batch fermentation procedure to be carried out after an in vitro gastrointestinal digestion. Therefore, this is an extension of the previous protocol described by Brodkorb et al. (2019)

The analysis of volatile organic compounds (VOCs) within breath for noninvasive disease detection and monitoring is an emergent research field that has the potential to reshape current clinical practice. However, adoption of breath testing has been limited by a lack of standardization. This protocol provides a comprehensive workflow for online and offline breath analysis using selected ion flow tube

Cellular heterogeneity is pervasive and of paramount importance in biology. Single-cell analysis techniques are indispensable for understanding the heterogeneity and functions of cells. Low-copy-number proteins (fewer than 1,000 molecules per cell) perform multiple crucial functions such as gene expression, cellular metabolism and cell signaling. The expression level of low-copy-number proteins of

Solid-state milling has emerged as an alternative, sustainable approach for preparing virtually all classes of compounds and materials. In situ reaction monitoring is essential to understanding the kinetics and mechanisms of these reactions, but it has proved difficult to use standard analytical techniques to analyze the contents of the closed, rapidly moving reaction chamber (jar). Monitoring by Raman

Measurements of neuronal activity across brain areas are important for understanding the neural correlates of cognitive and motor processes such as attention, decision-making and action selection. However, techniques that allow cellular resolution measurements are expensive and require a high degree of technical expertise, which limits their broad use. Wide-field imaging of genetically encoded indicators

Molecule-specific techniques such as MALDI and desorption electrospray ionization mass spectrometry imaging enable direct and simultaneous mapping of biomolecules in tissue sections in a single experiment. However, neurotransmitter imaging in the complex environment of biological samples remains challenging. Our covalent charge-tagging approach using on-tissue chemical derivatization of primary and

The nucleosome is the basic organizational unit of the genome. The folding structure of nucleosomes is closely related to genome functions, and has been reported to be in dynamic interplay with binding of various nuclear proteins to genomic loci. Here, we describe our high-throughput chromosome conformation capture with nucleosome orientation (Hi-CO) technology to derive 3D nucleosome positions with

The self-assembly of proteins into sophisticated multicomponent assemblies is a hallmark of all living systems and has spawned extensive efforts in the construction of novel synthetic protein architectures with emergent functional properties. Protein assemblies in nature are formed via selective association of multiple protein surfaces through intricate noncovalent protein–protein interactions, a challenging

As exemplified by the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there is a strong demand for rapid high-throughput isolation pipelines to identify potent neutralizing antibodies for prevention and therapy of infectious diseases. However, despite substantial progress and extensive efforts, the identification and production of antigen-specific antibodies remains labor-

Single-cell transcriptomics can profile thousands of cells in a single experiment and identify novel cell types, states and dynamics in a wide variety of tissues and organisms. Standard experimental protocols and analysis workflows have been developed to create single-cell transcriptomic maps from tissues. This tutorial focuses on how to interpret these data to identify cell types, states and other

The use of optogenetics to regulate neuronal activity has revolutionized the study of the neural circuitry underlying a number of complex behaviors in rodents. Advances have been particularly evident in the study of brain circuitry and related behaviors, while advances in the study of spinal circuitry have been less striking because of technical hurdles. We have developed and characterized a wireless

As engineered tissues progress toward therapeutically relevant length scales and cell densities, it is critical to deliver oxygen and nutrients throughout the tissue volume via perfusion through vascular networks. Furthermore, seeding of endothelial cells within these networks can recapitulate the barrier function and vascular physiology of native blood vessels. In this protocol, we describe how to

Gene editing by engineered nucleases has revolutionized the field of gene therapy by enabling targeted and precise modification of the genome. However, the limited availability of methods for clonal tracking of edited cells has resulted in a paucity of information on the diversity, abundance and behavior of engineered clones. Here we detail the wet laboratory and bioinformatic BAR-Seq pipeline, a strategy

Dopamine neuromodulation of neural synapses is a process implicated in a number of critical brain functions and diseases. Development of protocols to visualize this dynamic neurochemical process is essential to understanding how dopamine modulates brain function. We have developed a non-genetically encoded, near-IR (nIR) catecholamine nanosensor (nIRCat) capable of identifying ~2-µm dopamine release

Tissue clearing has become a powerful technique for studying anatomy and morphology at scales ranging from entire organisms to subcellular features. With the recent proliferation of tissue-clearing methods and imaging options, it can be challenging to determine the best clearing protocol for a particular tissue and experimental question. The fact that so many clearing protocols exist suggests there

Emerging evidence has demonstrated that RNA-RNA interactions are vital in controlling diverse biological processes, including transcription, RNA splicing and protein translation. RNA in situ conformation sequencing (RIC-seq) is a technique for capturing protein-mediated RNA-RNA proximal interactions globally in living cells at single-base resolution. Cells are first treated with formaldehyde to fix

Commensal bacteria from the human intestinal microbiota play important roles in health and disease. Research into the mechanisms by which these bacteria exert their effects is hampered by the complexity of the microbiota, the strict growth requirements of the individual species and a lack of genetic tools and resources. The assembly of ordered transposon insertion libraries, in which nearly all nonessential

Chemiresistors that are based on monolayer-capped metal nanoparticles (MCNPs) have been used in a wide variety of innovative sensing applications, including detection and monitoring of diagnostic markers in body fluids, explosive materials, environmental contaminations and food quality control. The sensing mechanism is based on reversible swelling or aggregation and/or changes in dielectric constant

3D correlative microscopy methods have revolutionized biomedical research, allowing the acquisition of multidimensional information to gain an in-depth understanding of biological systems. With the advent of relevant cryo-preservation methods, correlative imaging of cryogenically preserved samples has led to nanometer resolution imaging (2–50 nm) under harsh imaging regimes such as electron and soft

Existing protocols for full-length single-cell RNA sequencing produce libraries of high complexity (thousands of distinct genes) with outstanding sensitivity and specificity of transcript quantification. These full-length libraries have the advantage of allowing probing of transcript isoforms, are informative regarding single-nucleotide polymorphisms and allow assembly of the VDJ region of the T- and

Rigorous investigation of synaptic transmission requires analysis of unitary synaptic events by simultaneous recording from presynaptic terminals and postsynaptic target neurons. However, this has been achieved at only a limited number of model synapses, including the squid giant synapse and the mammalian calyx of Held. Cortical presynaptic terminals have been largely inaccessible to direct presynaptic

Cold atmospheric plasma (CAP) is a potential anticancer therapy. CAP has cytotoxic effects when applied either directly to cancer cell cultures or indirectly through plasma-conditioned liquids. This protocol describes how to treat adherent cultures of human cancer cell lines with CAP or plasma-conditioned medium and determine cell viability following treatment. The protocol also includes details on

Nature Protocols has traditionally published protocols for use in biological or biomedical research but has recently expanded its scope to include protocols for clinical practice.

Single-cell and single-nucleus sequencing techniques are a burgeoning field with various biological, biomedical and clinical applications. Numerous high- and low-throughput methods have been developed for sequencing the RNA and DNA content of single cells. However, for all these methods, the key requirement is high-quality input of a single-cell or single-nucleus suspension. Preparing such a suspension

Early prediction of patient outcomes is important for targeting preventive care. This protocol describes a practical workflow for developing deep-learning risk models that can predict various clinical and operational outcomes from structured electronic health record (EHR) data. The protocol comprises five main stages: formal problem definition, data pre-processing, architecture selection, calibration

Several essential components of the electron transport chain, the major producer of ATP in mammalian cells, are encoded in the mitochondrial genome. These 13 proteins are translated within mitochondria by ‘mitoribosomes’. Defective mitochondrial translation underlies multiple inborn errors of metabolism and has been implicated in pathologies such as aging, metabolic syndrome and cancer. Here, we provide

The global pandemic of coronavirus disease 2019 (COVID-19) highlights the shortcomings of the current testing paradigm for viral disease diagnostics. Here, we report a stepwise protocol for an RNA-extraction-free nano-amplified colorimetric test for rapid and naked-eye molecular diagnosis of COVID-19. The test employs a unique dual-prong approach that integrates nucleic acid (NA) amplification and

Faithful duplication of both genetic and epigenetic information is essential for all eukaryotic cells. DNA replication initiates from replication origins and proceeds bidirectionally but asymmetrically, with the leading strand being synthesized continuously and the lagging strand discontinuously as Okazaki fragments by distinct DNA polymerases. Unraveling the underlying mechanisms of chromatin replication

Determining chromatin-associated protein localization across the genome has provided insight into the functions of DNA-binding proteins and their connections to disease. However, established protocols requiring large quantities of cell or tissue samples currently limit applications for clinical and biomedical research in this field. Furthermore, most technologies have been optimized to assess abundant

The complex structure and function of a plant microbiome are driven by many variables, including the environment, microbe–microbe interactions and host factors. Likewise, resident microbiota can influence many host phenotypes. Gnotobiotic growth systems and controlled environments empower researchers to isolate these variables, and standardized methods equip a global research community to harmonize

Organs-on-chips have emerged as viable platforms for drug screening and personalized medicine. While a wide variety of human organ-on-a-chip models have been developed, rarely have there been reports on the inclusion of sensors, which are critical in continually measuring the microenvironmental parameters and the dynamic responses of the microtissues to pharmaceutical compounds over extended periods

The development of genetic tools allowed for the validation of the pro-aging and pro-disease functions of senescent cells in vivo. These discoveries prompted the development of senotherapies—pharmaceutical interventions aimed at interfering with the detrimental effect of senescent cells—that are now entering the clinical stage. However, unequivocal identification and examination of cellular senescence

Bacterial biofilms demonstrate high broad-spectrum adaptive antibiotic resistance and cause two thirds of all infections, but there is a lack of approved antibiofilm agents. Unlike the standard minimal inhibitory concentration assay to assess antibacterial activity against planktonic cells, there is no standardized method to evaluate biofilm inhibition and/or eradication capacity of novel antibiofilm

The genome is hierarchically organized into several 3D architectures, including chromatin loops, domains, compartments and regions associated with nuclear lamina and nucleoli. Changes in these architectures have been associated with normal development, aging and a wide range of diseases. Despite its critical importance, understanding how the genome is spatially organized in single cells, how organization