STAR Protocols

Protocol to establish rice root aphid colonies and experiments in greenhouse settings The rice root aphid (RRA) Rhopalosiphum rufiabdominalis (Sasaki) is a cosmopolitan species that attacks the root system of many plants grown in fields and greenhouses, including hydroponic production systems. Here, we present a protocol to establish consistent RRA colonies used to infest plants and to set up experiments in greenhouse settings. We describe steps for researching and understanding RRA colonies and establishing and maintaining the colony. We then provide suggestions for conducting s...

Protocol to establish rice root aphid colonies and experiments in greenhouse settings #protocol #starprotocols #cellpress

Protocol for the enhanced analysis of electrophysiological data from high-density multi-electrode arrays with nicespike and spikeNburst High-density multi-electrode arrays enable the recording of in vitro neuronal activity with exceptional spatial and temporal resolution. Here, we describe a protocol for analyzing these extensive datasets by using two complementary tools. The nicespike tool implements a full electrophysiological data analysis pipeline featuring graphics processing unit–accelerated spike sorting via template matching with Kilosort, enabling accurate identification of neuronal units across multiple electrodes. The...

Protocol for the enhanced analysis of electrophysiological data from high-density multi-electrode arrays with nicespike and spikeNburst #protocol #starprotocols #cellpress

Protocol for the expression, purification, and biochemical characterization of the innate immune sensor MDA5 MDA5 is one of the primary eukaryotic innate immune sensors of viruses, recognizing long double-stranded RNA (dsRNA). Here, we present procedures for the recombinant expression and purification of murine MDA5 from E. coli. We describe the steps to purify MDA5 in high yields for downstream experiments and procedures to determine the ATPase activity and RNA-binding properties of purified MDA5. These approaches can be used to produce disease-associated mutants of MDA5, to uncover the biochemical me...

Protocol for the expression, purification, and biochemical characterization of the innate immune sensor MDA5 #protocol #starprotocols #cellpress

Protocol combining RNA interference and regeneration assays in planarian embryos Planarians acquire regenerative abilities during late embryonic and juvenile development. Here, we present a protocol combining gene perturbation by RNA interference (RNAi) and regeneration assays in S. polychroa embryos. We describe steps for embryo staging, amputation, double-stranded RNA soaking, and phenotype analysis. This protocol is adaptable for use with embryonic stages amenable to ex vivo culturing and with other embryo-producing flatworm species. For complete details on the use and ex...

Protocol combining RNA interference and regeneration assays in planarian embryos #protocol #starprotocols #cellpress

Protocol for imaging-based quantification of RNAPII clearance during transcription-coupled DNA repair Elongating RNA polymerase II (RNAPII) stalls at transcription-blocking lesions in the DNA template strand and is removed by transcription-coupled DNA repair (TCR) factors. Here, we present a protocol to measure RNAPII clearance during TCR at sites of localized UV-induced DNA damage in adherent cells. We describe how to induce local UV damage and visualize the damaged area and chromatin-bound RNAPII levels using immunofluorescence staining. This approach can quantify the clearance of DNA damage-s...

Protocol for imaging-based quantification of RNAPII clearance during transcription-coupled DNA repair #protocol #starprotocols #cellpress

Protocol to investigate gene expression heterogeneity in cyanobacteria using mRNA CARD-FISH Here, we present a protocol for visualizing gene expression in the filamentous cyanobacterium Trichodesmium and the single-celled species Synechocystis and Cyanothece using the catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) technique. We describe steps for fixation, agarose coating, enzymatic permeabilization, and sample handling. This protocol is broadly applicable to cyanobacteria, and the detection of rbcL mRNA in Trichodesmium, used as an example, supports its u...

Protocol to investigate gene expression heterogeneity in cyanobacteria using mRNA CARD-FISH #protocol #starprotocols #cellpress

Protocol to clone unknown flanking genomic region using SWPOP-PCR for genome walking Genome walking is a molecular technique for mining unknown flanking DNAs, significantly advancing fields related to biology. We here detail a genome-walking protocol for stepwise partially overlapping primer-based PCR (SWPOP-PCR). We specify the rules for designing walking primers and gene-specific primers. We also delineate the three-stage workflow of SWPOP-PCR for cloning target DNA, as well as the subsequent purification, sequencing, and analysis of target amplicon. For complete details on th...

Protocol to clone unknown flanking genomic region using SWPOP-PCR for genome walking #protocol #starprotocols #cellpress

Protocol to study human Kv1.2 potassium channel pathogenic sequence variants using two-electrode voltage-clamp technique The abietane diterpenoid pisiferic acid (PA) from conifer Chamaecyparis pisifera is a pan-rescuer of human Kv1.2 channel pathogenic loss-of-function (LOF) sequence variants. Here, we present a protocol for the study of Kv1.2 channel sequence variants using the Xenopus laevis oocyte expression system and two-electrode voltage-clamp (TEVC) electrophysiology. We describe steps for studying mutant Kv1.2, wild-type Kv1.2, and wild-type Kv1.1 cRNA combinations in Xenopus laevis oocytes; performing TEV...

Protocol to study human Kv1.2 potassium channel pathogenic sequence variants using two-electrode voltage-clamp technique #protocol #starprotocols #cellpress

Protocol for investigating mitochondrial structure, function, and metabolism in human cervical cancer cells Mitochondria regulate a variety of biological activities, including metabolism, oxidative stress, and cell death. Here, we present a protocol for the investigation of mitochondrial structure, function, and metabolism in human cervical cancer cells. We describe steps for staining and visualizing mitochondria using confocal microscopy to assess morphology, mass, membrane potential, calcium, reactive oxygen species (ROS), and lipid droplet accumulation. We then detail procedures for isolating mitoc...

Protocol for investigating mitochondrial structure, function, and metabolism in human cervical cancer cells #protocol #starprotocols #cellpress

Protocol to measure insulin secretion dynamics from single mouse islets using a microfluidic fluorescence anisotropy immunoassay The dynamics of glucose-stimulated insulin secretion (GSIS) from islets offer valuable insight into β cell metabolism. Here, we present a protocol for using a microfluidic device (i.e., InsC-chip) to measure the temporal dynamics of GSIS from individual mouse islets. We describe the steps for fabricating polydimethylsiloxane (PDMS)-based chips through soft lithography. We then present detailed procedures for employing the InsC-chip to measure insulin secretion from either four or eight individua...

Protocol to measure insulin secretion dynamics from single mouse islets using a microfluidic fluorescence anisotropy immunoassay #protocol #starprotocols #cellpress

Protocol for fabricating a starfish-inspired magnetoelastic generator array This protocol outlines the fabrication and application of a starfish-inspired magnetoelastic generator (MEG) array for ocean wave energy harvesting and autonomous hydrogen (H2) production. We detail the procedures for device design, frame and MEG fabrication, magneto-mechanical coupling (MC) layer characterization, and array assembly. We also describe procedures for evaluating electrical performance, converting AC to DC output, and enabling MEG-driven on-site water-splitting, offering a comprehe...

Protocol for fabricating a starfish-inspired magnetoelastic generator array #protocol #starprotocols #cellpress

Protocol for a mouse tubuloid model of myoglobinuric acute kidney injury Myoglobinuric acute kidney injury (AKI) occurs as a result of rhabdomyolysis. Here, we present a protocol based on mouse primary renal tubular epithelial organoids, or “tubuloids”, to model myoglobinuric AKI in vitro. We describe how to establish and maintain the culture of mouse tubuloids and how to trigger myoglobin-induced injury. Moreover, we provide instructions on how to quantify tubuloid damage. For complete details on the use and execution of this protocol, please refer to Zhou et al.1

Protocol for a mouse tubuloid model of myoglobinuric acute kidney injury #protocol #starprotocols #cellpress

Bridging mesoscopic and microscopic scales in multiple sclerosis: Post mortem brain block multi-contrast 9.4T MRI and histology quantification Combining ex vivo MRI mesoscopic and histopathology microscopic assessments of tissue microstructure forms a key approach in multiple sclerosis (MS) research for elucidating disease pathogenesis. We present a protocol that enables reproducible, scalable, and robust integration of MRI and histology data in MS brains. We describe steps for (1) obtaining high-resolution multi-contrast 9.4T MRI of postmortem brain blocks and (2) the usage of a custom, open-source image processing interface for robus...

Bridging mesoscopic and microscopic scales in multiple sclerosis: Post mortem brain block multi-contrast 9.4T MRI and histology quantification #protocol #starprotocols #cellpress

Protocol for culturing olfactory epithelium organoids supporting neuronal differentiation The olfactory epithelium (OE) contains stem cells capable of generating olfactory sensory neurons throughout life, making it a valuable model for studying epithelial neurogenesis. We present a protocol to develop a highly robust 3D mouse organoid model from OE cells. We describe a workflow for dissecting murine OE and subsequent organoid culturing. We also provide guidance on how to perform immunostaining of the organoids. This protocol has been validated for mice and does not require fluorescen...

Protocol for culturing olfactory epithelium organoids supporting neuronal differentiation #protocol #starprotocols #cellpress

Protocol for dynamic high-throughput cell death screening of primary phagocytes following microplastic and nanoplastic exposure Micro- and nanoplastics (MNPs) can cross epithelial barriers of the lung and/or intestine into the bloodstream. In the body, phagocytes will be exposed to plastic particles, but they are incapable of degrading them. Here, we present a protocol for high-throughput cell death screening of primary phagocytes following MNP exposure. We describe steps for isolating primary phagocytes, plating these with MNPs, and time-lapse imaging. Further, we explain detailed procedures for image analysis using the...

Protocol for dynamic high-throughput cell death screening of primary phagocytes following microplastic and nanoplastic exposure #protocol #starprotocols #cellpress

Protocol for genetic discovery and fine-mapping of multivariate latent factors from high-dimensional traits High-dimensional traits, like blood cell traits, are often analyzed using univariate genetic analysis approaches, ignoring trait relationships. Here, we present a protocol for using the flashfmZero software for analyses of latent factors that capture variation in observed traits generated by shared underlying biological mechanisms. We describe steps for calculating genome-wide association study (GWAS) summary statistics of latent factors from GWAS of observed traits, allowing for missing trait m...

Protocol for genetic discovery and fine-mapping of multivariate latent factors from high-dimensional traits #protocol #starprotocols #cellpress

Protocol for quantifying the thickness and puncture resistance properties of solitary bee cocoons using Osmia lignaria as a model Solitary bee cocoons serve as protective barriers against parasitoids, yet their puncture resistance properties remain understudied. Here, we present a protocol for measuring puncture resistance in Osmia lignaria cocoons using custom 3D-printed fixtures. We describe steps for determining bee sex, preparing cocoon segments, measuring segment thickness, testing puncture resistance using 27.5-gauge sharp needles on an MTS Synergie 100 mechanical testing instrument, and analyzing the data. The proto...

Protocol for quantifying the thickness and puncture resistance properties of solitary bee cocoons using Osmia lignaria as a model #protocol #starprotocols #cellpress

Protocol for RNA-seq library preparation from low-volume total RNA by RNA/cDNA hybrid tagmentation RNA sequencing (RNA-seq) is a widely used and powerful technique for studying gene expression. Among the various protocols, SHERRY (sequencing hetero RNA-DNA-hybrid) profiles polyadenylated RNAs by direct tagging of RNA/DNA hybrids and offers a robust and economical way for gene expression quantification. Here, we present a detailed protocol for standard SHERRY library preparation from 200 ng of total RNA. We describe steps of RNA purification, reverse transcription, hybr...

Protocol for RNA-seq library preparation from low-volume total RNA by RNA/cDNA hybrid tagmentation #protocol #starprotocols #cellpress

Protocol for decoding immune predictors of response to immunotherapy through pan-cancer multiomics analysis Here, we present a protocol for analyzing T cell dynamics in multiple cancers through single-cell RNA sequencing (scRNA-seq), single-cell immune profiling, and mass cytometry/cytometry by time-of-flight (CyTOF). We describe steps for performing gene-regulatory network analysis to identify key transcription factors and cell-cell interaction analysis to explore immune signaling. This protocol facilitates the identification of T cell subsets associated with immune checkpoint...

Protocol for decoding immune predictors of response to immunotherapy through pan-cancer multiomics analysis #protocol #starprotocols #cellpress

Protocol for mapping murine transcription factor interactomes and composite motifs combining affinity purification mass spectrometry and ChIP-seq Mass spectrometry (MS)-based approaches have significantly advanced our ability to study protein interaction networks in an unbiased manner. Here, we present a protocol that uses affinity purification (AP)-MS to identify interaction partners of a biotinylated transcription factor of interest, isolated from primary murine T cells. The resulting interactome data are integrated with motif analyses from chromatin immunoprecipitation sequencing (ChIP-seq) experiments. This com...

Protocol for mapping murine transcription factor interactomes and composite motifs combining affinity purification mass spectrometry and ChIP-seq #protocol #starprotocols #cellpress

Scanning probe microscopy for rheological analysis of biomolecular condensates Biomolecular condensates are membrane-less phase-separated assemblies that play key roles in cellular organization. Here, we present a protocol to characterize the full range of rheological properties of condensate droplets, from liquid to solid states, using scanning probe microscopy. We describe steps for preparing condensates, cantilever calibration, and condensate measurement. We then detail analysis procedures required to derive the complex shear modulus. For complet...

Scanning probe microscopy for rheological analysis of biomolecular condensates #protocol #starprotocols #cellpress

Protocol for an automated virtual screening pipeline including library generation and docking evaluation Here, we present a protocol for an automated virtual screening pipeline. We describe steps for generating compound libraries for computational docking including Food and Drug Administration (FDA)-approved drugs, setting up the receptor and grid box, and docking a library of compounds. We then detail procedures for ranking docking results. This protocol offers scripts for Unix-like systems, lowering the access barrier for researchers interested in structure-based drug disc...

Protocol for an automated virtual screening pipeline including library generation and docking evaluation #protocol #starprotocols #cellpress

Covalent anchoring of tethered ligands to chemogenetic handles for targeted in vivo neuropharmacology Covalent anchoring of tethered ligands to chemogenetic handles (CATCH) is a chemogenetic approach that enables sustained, localized, and pharmacologically specific receptor antagonism. Here, we present a protocol for designing and applying CATCH in mice. We describe how to integrate CATCH with ex vivo and in vivo electrophysiological recordings, as well as detail procedures for combining CATCH with behavioral assays. CATCH enables the specific control of neurotransmitter receptor function, offer...

Covalent anchoring of tethered ligands to chemogenetic handles for targeted in vivo neuropharmacology #protocol #starprotocols #cellpress

FlowLITE: A protocol to characterize and quantify total antibody isotypes in human plasma using flow cytometry FlowLITE is a flow cytometry bead-based assay that enables multiplexing and high-throughput quantification of all antibody isotypes from humans, mice, or other species, using small volumes of any fluid specimen. Here, we detail a FlowLITE protocol to quantify and characterize antibody light-chain and isotype expression in human plasma. We describe steps for titrating the capture and reveal antibodies, multiplexing assay beads, and staining samples. We then describe proced...

FlowLITE: A protocol to characterize and quantify total antibody isotypes in human plasma using flow cytometry #protocol #starprotocols #cellpress

Protocol for preparation of mouse synovium for flow cytometry and RNA-seq The mouse synovium can be used to model the sterile inflammation observed in rheumatoid arthritis and osteoarthritis. Here, we present a protocol for inducing arthritis and harvesting and preparing a single-cell suspension from cells of interest from the mouse synovium in steady state or inflammation, with or without intravascular labeling. The prepared single-cell suspension can then be used for downstream analyses, including flow cytometry, fluorescence-activated cell s...

Protocol for preparation of mouse synovium for flow cytometry and RNA-seq #protocol #starprotocols #cellpress

Protocol to generate stable knockout lines in the human-parasitic nematode Strongyloides stercoralis A major limitation to the study of gene function in parasitic nematodes was the inability to make stable mutant lines. Here, we present a protocol for generating stable knockout lines in the human-parasitic nematode Strongyloides stercoralis. We describe steps for generating CRISPR components and microinjecting them into worms. We also detail procedures for identifying potential gene disruptions and propagating mutants by host passage in gerbils to generate stable homozygous knockout lines. This...

Protocol to generate stable knockout lines in the human-parasitic nematode Strongyloides stercoralis #protocol #starprotocols #cellpress

Protocol for autofluorescence removal in microglia by photobleaching in free-floating immunofluorescent staining of mouse brain tissue Autofluorescence in brain tissue poses a challenge in immunofluorescent staining by obscuring antibody-labeled proteins, mainly within microglia. Here, we describe a cost-effective protocol for removing autofluorescence in mouse brain sections by photobleaching. We outline steps to collect and section brain tissue, apply a photobleaching step using a light-emitting diode (LED), and perform immunofluorescent staining with primary and secondary antibodies. This protocol enables clearer visualizati...

Protocol for autofluorescence removal in microglia by photobleaching in free-floating immunofluorescent staining of mouse brain tissue #protocol #starprotocols #cellpress

Protocol for the generation of three-dimensional micropatterned neuroepithelial tissues using hPSCs, bioprinting, and matrix scaffolds Micropatterning technology that spatially guides the self-assembly of human pluripotent stem cells (hPSCs) into neural tissues offers enhanced fidelity to investigate early neurodevelopment and disease mechanisms. Here, we present a protocol to produce micropatterned and scaffolded neuroepithelial tissues (scNETs) from hPSCs, leveraging three-dimensional (3D) bioprinting to deposit extracellular matrix (ECM) droplets with defined geometries. We describe steps for bioprinting of ECM micropatterns...

Protocol for the generation of three-dimensional micropatterned neuroepithelial tissues using hPSCs, bioprinting, and matrix scaffolds #protocol #starprotocols #cellpress

Protocol to quantify mechanical properties of cells using optical tweezers Mechanical properties of cells determine their ability to deform when subjected to force, which is crucial in many biological processes. Here, we present a protocol to quantify these properties on living cells by applying piconewton-range forces with optically trapped microspheres. We describe steps for sample preparation of both adherent and suspended cells, calibration of optical trap stiffness, and generation of force-deformation curves. We next detail how to quantify cellular mechanical prop...

Protocol to quantify mechanical properties of cells using optical tweezers #protocol #starprotocols #cellpress

Protocol for creating a gene dictionary for organelle genomes using the Gene Dictionary Tool Here, we present a protocol for creating a gene dictionary for fungal mitochondrial genomes using the Gene Dictionary Tool. Through a Python Command Line Interface (CLI), the user identifies what annotations are missing in the inputted dictionary. Via two Jupyter Notebooks, the user builds a gene dictionary based on attributes retrieved from inputted GFF3 files. The final output, a .gdict file, is findable, accessible, interoperable, and reusable (FAIR). This protocol can be adapted to create a ...

Protocol for creating a gene dictionary for organelle genomes using the Gene Dictionary Tool #protocol #starprotocols #cellpress