Transcranial magnetic stimulation (TMS) perturbs specific brain regions and, combined with electroencephalography (EEG), enables the assessment of act…

Traditionally, midbody remnants (MBRs) are isolated from cell culture medium using ultracentrifugation, which is expensive and time consuming. Here, w…

Expansion microscopy (ExM) is a tissue-swelling technique that enables super-resolution imaging through a specialized preparation process that physically expands stained biomolecules within a fixed sample. Here, we detail a protocol to apply the 4× ExM technique to neuromuscular junctions (NMJs) from both human and mouse muscle preparations. We describe steps for muscle fixation, microdissection, staining, gelation, and digestion. We then detail procedures for expansion, mounting, imaging, analy...

Here, we present a protocol to study epidermal growth factor (EGF) receptor (EGFR) or transferrin trafficking in mammalian cells. We describe steps for using fluorescent ligands or antibodies, confocal imaging, and quantitative analysis to track their…

Here, we present a protocol for polysome profiling in differentiating adipocytes from the mouse stromal vascular fraction. We describe steps for lysate preparation, ultracentrifugation through sucrose gradients, mRNA isolation, RNA sequencing, and motif enrichment. This protocol enables the analysis of actively translating mRNAs and translational gene regulation by isolating polysome-bound mRNA, revealing insights into protein synthesis during adipogenesis or stimuli responses. Applications incl...

STAR Protocols is an open access, peer-reviewed journal from Cell Press. We offer structured, transparent, accessible, and repeatable step-by-step experimental and computational protocols from all are...

When translation elongation is hindered, stalled ribosomes can collide with trailing ribosomes and form ribosome collision complexes also known as disomes. Disomes are sensed in the cell to trigger signaling events, and therefore it is important to determine their frequency and distribution across transcripts. Here, we provide the protocol for Disome-seq in Saccharomyces cerevisiae (yeast) cells to enable transcriptome-wide detection of disomes. We describe the steps for yeast growth and isolati...

Artificial plastic substrates (APSs) consist of four plastic polymers that work as traps for pathogenic bacteria and facilitate their sampling and detection. Here, we present a protocol for sampling antibiotic-resistant pathogenic bacteria in aquatic ecosystems using APSs. We describe steps for selecting plastic polymers, followed by their assembly, submergence in aquatic environments, and collection for analysis. This protocol can also help explore plastic’s role as a new route for disease expo...

Artificial intelligence (AI) has revolutionized structural biology but must be applied reliably. Here, we present an approach for derivation of distance restraints from AlphaFold structure predictions to aid in automated nuclear Overhauser effect (NOE) assignment during solution NMR structure determination. We describe steps for selecting reliable AlphaFold structure predictions, determination of atom distances, and generation of high-confidence distance restraints. This protocol can expedite so...

Here, we present a protocol for using SIMS (scalable, interpretable machine learning for single cell) to transfer cell type labels in single-cell RNA sequencing data. This protocol outlines data preparation, model training with labeled data or inference using pretrained models, and methods for visualizing, downloading, and interpreting predictions. We provide stepwise instructions for accessing SIMS through the application programming interface (API), GitHub Codespaces, and a web application. Fo...

Non-coding variants can alter transcription factor (TF)-DNA binding and dysregulate gene expression. Here, we present a protocol for evaluating the impact of non-coding variants on TF binding and gene expression. We describe steps for expressing and purifying DNA-binding proteins, preparing DNA probes and reporter constructs, and measuring changes in TF-DNA binding affinity. Finally, we provide instructions for quantification and statistical analysis. For complete details on the use and executio...

Protein tagging with CRISPR-Cas9 enables the investigation of protein function in its native environment but is limited by low homology-directed repair (HDR) efficiency. Here, we present a protocol for fast antibiotic resistance-based gene editing with CRISPR-Cas9 (FAB-CRISPR), which streamlines N/C-terminal tagging using an antibiotic resistance cassette for rapid selection and enrichment of gene-edited cells. We describe in detail guide RNA and HDR donor plasmid cloning, transfection of editin...

Quantifying the phosphorylation levels of proteins involved in bacterial signaling pathways is essential for understanding their role in stress adaptation and infection processes. Here, we present a protocol for the semi-quantitative assessment of the phosphorylation level of the transcriptional regulator CpxR during the infection process using Phos-tag gel. We describe steps for Dickeya dadantii infection of chicory leaves, followed by bacterial extraction, rapid acidic cell lysis, and subseque...

A valuable and widely utilized approach to study gene regulatory networks is through transgenic lines. However, detailed information of transgene insertion sites is often not available, complicating the interpretation of experimental results involving transgenes. Here, we present a protocol for mapping the insertion sites of zebrafish Tol2 transgenes, using the recently developed tagmentation-based TransTag technique. We describe detailed steps for preparing sequencing libraries and analyzing ra...

Neuronal ensembles are groups of neurons with coordinated activity that are related to a specific brain function. Here, we present a protocol to identify and compare neuronal ensembles from neuronal activity data obtained through two-photon microscopy or electrophysiological recordings using a unified graphical user interface (Ensembles Comparison and Recognition [ENCORE]). We describe steps for installing ENCORE, data loading, neuronal ensemble identification, inspecting results, and comparison...

Cell fractionation enables the study of compartment-specific RNA processing events in organisms with complex cell walls. Here, we present a protocol for isolating nuclear and cytosolic fractions from Saccharomyces cerevisiae. We describe spheroplast formation using zymolyase, followed by subcellular fractionation via a sucrose density gradient. Finally, we detail the extraction of high-quality RNA from these fractions. This approach overcomes cell wall barriers to allow the analysis of ribosome ...

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...