Single-cell RNA sequencing (scRNA-seq) is a powerful technique for exploring cellular heterogeneity and host–pathogen interactions. This protocol details the Zika virus (ZIKV)-targeted scRNA-seq workflow for preparing high-quality single-cell suspensions from the whole brain tissues of neonatal mice, high-quality single-cell sorting, cDNA reverse transcription, amplification, ZIKV enrichment and host transcriptome library preparation, and sequencing dataset integration in downstream analysis to complete the quantification of ZIKV RNA in individual cells.Key features• Preparation of high-quality single-cell suspensions from the whole brain tissues of neonatal mice.• ZIKV-specific magnetic beads for using the ZIKV and host cell RNA capture.• ZIKV enrichment and host transcriptome library construction, providing a framework for quantifying viral load within individual cells.• Integration of viral enrichment and host transcriptomic datasets enables the visualization and quantification of ZIKV at single-cell resolution.

Human tissue samples represent the gold standard for obtaining clinically relevant and translatable insight into disease processes that in vitro systems cannot fully reproduce. However, patient-derived samples are often limited in size and availability, limiting the number of downstream assays that can be performed. To maximize the use of invaluable human samples, we present a protocol for the tandem extraction of high-quality RNA and protein from the same tissue section. This method has been optimized for 15–30 mg tissue sections, enabling more experimental conditions and technical replicates, while minimizing intrasample variability associated with heterogeneous tissues. This protocol also avoids potentially hazardous solvents present in phenol-chloroform-based methods such as TRIzol, providing a safer and more accessible workflow without compromising biomolecule integrity. This protocol was developed and validated using atherosclerotic plaque tissue from carotid endarterectomy, a very challenging tissue type to work with due to extensive calcification, necrosis, and limited surgical availability. We have also validated this method using mouse aortic tissue and cultured THP-1 cells, demonstrating its versatility across sample input types. As this protocol relies on standard column-based RNA extraction kits and commonly available reagents for protein precipitation and extraction, this methodology is widely accessible and easy to implement as a standard, streamlined workflow.Key features• Advances ex vivo models of human disease (e.g., atherosclerosis) by maximizing the use of patient-derived tissue samples.• Minimizes patient variability by generating directly comparable RNA and protein readouts.• Minimizes the number of patient samples required and maximizes the output from each sample.• Stabilizes RNA integrity from tissue without impacting the quality of proteins derived from the same sample.

ADGRL4 is an adhesion G protein-coupled receptor (aGPCR) implicated in tumour progression in multiple malignancies. We recently determined the first cryo-EM structure of active-state ADGRL4, revealing its weak coupling to the heterotrimeric G protein Gq and providing insights into its activation mechanism. Here, we describe a complete modular workflow for purifying active-state ADGRL4 over 2–3 days using a multifunctional tagging strategy incorporating multiple orthogonal detection, purification, and cleavage tags at the N-terminus as well as a tethered mini-Gq at the C-terminus. This configuration enhanced receptor cell-surface expression and stability and allowed different purification strategies to be tested during the development of the purification protocol. Although developed and optimised for ADGRL4, this approach is readily transferable to other weakly coupling aGPCRs or GPCRs where complex stability is a limiting factor for structural analysis.Key features• Complete workflow for purifying active-state ADGRL4 for cryo-EM analysis.• Modular, multifunctional N-terminal tagging strategy supporting multiple orthogonal purification and detection methods without any negative effect on cell surface expression levels.• Tethered mini-Gq increases stability and receptor cell-surface expression.• Modular purification tagging configuration provides freedom to change purification methodologies without having to perform additional receptor engineering or cloning.

RNA-binding proteins (RBPs) have pleiotropic roles in modulating the physiology of both eukaryotic and prokaryotic cells, enabling them to adapt to environmental variations. The importance of RBPs has led to the development of a variety of methods aiming to identify them. However, most of these approaches have primarily been implemented and optimized in eukaryotic systems. To both uncover novel RBPs involved in Bacillus subtilis sporulation and capture their RNA-binding ability dynamically, we adapted the orthogonal organic phase separation technique (OOPS), which had previously been used in Escherichia coli to reveal its RNA-binding proteome (RBPome). We optimized the UV cross-linking process used to stabilize RNA–protein interactions in vivo and the bacterial lysis process to overcome the robust cell wall of Gram-positive sporulating cells. RNA–protein complexes are then recovered after phase separation steps using guanidinium thiocyanate–phenol–chloroform, and RNA-associated proteins are identified and label-free-quantified by liquid chromatography–mass spectrometry. Collecting samples at various time points during sporulation further enables tracking the dynamics of the RBPome. In addition to being applicable to bacteria and requiring minimal starting material, this method has provided a comprehensive map of the RBPome during sporulation, refining the roles of known factors and revealing new players.Key features• The high-throughput method OOPS, developed in [1], was successfully applied to both sporulating and vegetative cells of a Gram-positive bacterium to specifically purify the RBPome.• OOPS allowed tracking of RBPome remodeling dynamics (both RBP abundance and RNA binding ability) across different stages of B. subtilis sporulation.• OOPS enabled the identification of novel RBPs in the context of sporulation, revealing potential new players in RNA-mediated regulation.

RNA-binding protein (RBP)–RNA interactions are fundamental for gene regulation and cellular homeostasis. Ataxin-2 is an RBP that has been shown to play an instrumental role in pathophysiological processes by binding to mRNA. Methods such as RNA immunoprecipitation (RIP), cross-linking immunoprecipitation (CLIP), and their variants can be used to study the interactions between Ataxin-2 and its targets, although their high sample requirements and labor-intensive workflows can limit their widespread use. RNA editing-based approaches, such as targets of RBPs identified by editing (TRIBE), provide effective alternatives. TRIBE enables transcriptome-wide identification of RBP targets by inducing site-specific adenosine-to-inosine (A-to-I) editing, which is subsequently detected through high-throughput RNA sequencing in both in vivo and in vitro systems. Compared to in vivo models, cell lines offer a rapid and flexible experimental design. Drosophila S2 cells are a commonly used insect cell line to investigate RNA–protein dynamics and serve as a versatile platform for studying RBP function. Here, we describe a protocol used for identifying RNA targets of Ataxin-2, a versatile RBP involved in post-transcriptional and translational regulation, in S2 cells using TRIBE. This method allows rapid, efficient, and reliable identification of Ataxin-2-associated RNA targets and can be readily applied to other RBPs.Key features• Streamlined workflow for identifying RNA targets of RBPs using TRIBE in Drosophila S2 cells.• TRIBE highlights the sites and nature of interactions between RBPs and RNA.• TRIBE overcomes the limitations of conventional RBP–RNA interaction studies like RIP, CLIP, and their variants.

Evaluating single-domain antibody cooperativity is essential for developing potent, escape-resistant antiviral biologics. Here, we present a protocol that reproducibly quantifies functional synergy between neutralizing nanobody pairs in standardized viral infectivity assays. Controlled automated liquid handling prepares two-dimensional concentration matrices, minimizing pipetting variance and systematic error. Neutralization data are fitted using quantitative models that independently estimate potency, cooperativity, and efficacy to distinguish additive, synergistic, and antagonistic effects between nanobody pairs. Replicated measurements enable statistically interpretable parameter estimates, supporting robust evaluation of combinatorial nanobody therapeutics with commonly available equipment and open-source analysis tools. This framework is broadly applicable to assessing cooperative effects among other classes of binding or inhibitory molecules, facilitating systematic discovery of synergistic combinations.Key features• Understanding functional synergistic effects between antiviral nanobodies advances their rational therapeutic design.• Performing nanobody synergy experiments with an automated liquid handling workflow enables high-throughput and highly reproducible analysis of nanobody cooperativity.• Evaluating a two-dimensional matrix of nanobody concentrations using the MuSyC model provides the resolution needed to understand how one nanobody can be influenced by another.• This protocol integrates experimental, computational, and visualization procedures into a unified framework to identify and characterize synergistic antiviral nanobody combinations.

Membrane-less organelles play essential roles in both physiological and pathological processes by compartmentalizing biomolecules through phase separation to form dynamic hubs. These hubs enable rapid responses to cellular stress and help maintain cellular homeostasis. However, a straightforward and efficient method for detecting and illustrating the distribution and diversity of RNA species within membrane-less organelles is still highly sought after. In this study, we present a detailed protocol for in situ profiling of RNA subcellular localization using Target Transcript Amplification and Sequencing (TATA-seq). Specifically, TATA-seq employs a primary antibody against a marker protein of the target organelle to recruit a secondary antibody conjugated with streptavidin, which binds an oligonucleotide containing a T7 promoter. This design enables targeted, in situ reverse transcription of RNAs with minimal background noise, a key advantage further refined during data analysis by subtracting signals obtained from a parallel IgG control experiment. The subsequent T7 RNA polymerase-mediated linear amplification ensures high-fidelity RNA amplification from low-input material, which directly contributes to optimized sequencing metrics, including a duplication rate of no more than 25% and a mapping ratio of approximately 90%. Furthermore, the modular design of TATA-seq provides broad compatibility with diverse organelles. While initially developed for membrane-less organelles, the protocol can be readily adapted to profile RNA in other subcellular compartments, such as nuclear speckles and paraspeckles, under both normal and pathogenic conditions, offering a versatile tool for spatial transcriptomics. Key features • This protocol provides subcellular spatial resolution by targeting and sequencing RNA from specific membrane-less organelles. • A T7-based linear amplification step ensures high sensitivity and yield from low-input samples (<10,000 cells). • The method is adaptable for profiling diverse organelles under various biological conditions.

The cellular compartments of eukaryotic cells are defined by their specific protein compositions. Different strategies are used for the identification of the subcellular proteomes, such as fractionation by differential centrifugation of cellular extracts. The localization of mitochondrial proteins is particularly challenging, as mitochondria consist of two membranes of different protein composition and two aqueous subcompartments, the intermembrane space (IMS) and the matrix. Previous studies identified subcompartment-specific proteomes by using combinations of hypotonic swelling and protease digestion followed by mass spectrometry. Here, we present an alternative, more unbiased method to identify the proteomes of mitochondrial subcompartments by use of an improved ascorbate peroxidase (APEX2) that is targeted to the IMS and the matrix. This method allows the subcompartment-specific labeling of proteins in mitochondria isolated from cells of the baker's yeast Saccharomyces cerevisiae, followed by their purification on streptavidin beads. With this method, the proteins located in the different mitochondrial subcompartments of yeast cells can be efficiently and comprehensively identified. Key features • Coverage of ~75% of previous combined annotated mitochondrial proteome studies with high confidence in sub-localization probabilities. • Provides detailed steps from starting culture to MS sample preparation, including the isolation of mitochondria. • Allows for easy adaptations to compare different conditions and treatments. • The whole experiment requires at least five days to complete.

This protocol describes a reproducible workflow for modeling in vitro impact-induced traumatic brain injury (TBI) using a mechanical stretch system applied to differentiated SH-SY5Y human neuroblastoma cells cultured on polydimethylsiloxane (PDMS) substrates. The protocol integrates three primary components: (1) fabrication and surface modification of deformable PDMS chambers to support cellular adhesion, (2) partial differentiation of SH-SY5Y cells using retinoic acid, and (3) induction of controlled mechanical strain to simulate mild to moderate TBI. The stretch-induced injury model enables quantitative assessment of cellular viability and recovery following mechanical insult. This approach provides a versatile platform for studying cellular and molecular mechanisms of TBI, screening neuroprotective compounds, and exploring mechanobiological responses in neural cells under controlled strain magnitudes and rates.Key features• Provides reproducible in vitro modeling of traumatic brain injury in differentiated SH-SY5Y neurons via controlled mechanical stretch.• Combines PDMS chamber fabrication and polydopamine coating to generate deformable, biocompatible, neuron-adhesive substrates for mechanobiology assays.• Flexible platform for testing neuroprotective compounds and investigating mechanotransduction pathways under physiologically relevant stretch conditions.

Neuronal survival in vitro is usually used as a parameter to assess the effect of drug treatments or genetic manipulation in a disease condition. Easy and inexpensive protocols based on neuronal metabolism, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), provide a global view of protective or toxic effects but do not allow for the monitoring of cell survival at the single neuronal level over time. By utilizing live imaging microscopy with a high-throughput microscope, we monitored transduced primary cortical neurons from 7–21 days in vitro (DIV) at the single neuronal level. We established a semi-automated analysis pipeline that incorporates data stratification to minimize the misleading impact of neuronal trophic effects due to plating variability; here, we provide all the necessary commands to reproduce it.Key features• The protocol enables monitoring of primary cortical neuron survival from DIV 7 to 21 in 96-well plates following various cellular treatments.• It provides single-cell and real-time imaging resolution, enabling the identification of small changes in viability over time.• It provides a detailed description of semi-automated neuronal detection over time.• It relies on data stratification based on the neuronal starting number, which helps reduce the impact of neuronal trophic effects due to plating variability.• It has been used to assess the effect of glial extracellular vesicles on cortical neurons, as reported in [1].