STAR Protocols

The bacterial three-hybrid (B3H) assay is a powerful genetic tool for detecting interactions between RNA and RNA-binding proteins (RBPs) and assessing the consequences of RBP mutations. This transcription-based system connects the strength of an RNA-protein interaction to the expression of a lacZ reporter gene in Escherichia coli cells. This in vivo approach allows researchers to dissect RNA-protein interactions within a cellular environment, bypassing the need for biochemical purification of RNAs or proteins. This chapter details a three-day protocol for generating quantitative B3H data. Since a significant challenge in B3H assays is RNA misfolding, we describe a recently optimized set of B3H constructs that mitigates this issue by isolating bait RNAs as discrete folding units.

O_LIThe effect of some bioactive compounds on living organisms is dependent on their concentration and gradients, as is the case of hormones and signalling peptides, determining cell identity, activity and organism development. C_LIO_LIThere are a handful of methods that allow to produce spatially confined peaks of concentration local application of biochemicals on plants, such as agar blocks and microinjection, but they lack in precision, throughput and/or simplicity. C_LIO_LIWe developed the MicroTron, a microfluidics-based method specifically for filamentous organisms or life cycle stages, like the moss plant Physcomitrium patens protonemata, that serves as a platform for the application of chemicals on single cells and study the cell response. C_LIO_LIWe show how chemical applications could be performed on cells, either on the side or apically with dyes and hormones, targeting the cell wall, cell membrane, cytosol and nucleus. C_LIO_LITreatments could be applied on single filaments and with a precision of up to single cells in optimal conditions. C_LIO_LIThis method could be used to study live responses to chemicals with high spatiotemporal resolution. C_LI

Integrin v{beta}8 is a major activator of latent transforming growth factor-{beta} (TGF-{beta}) and an emerging therapeutic target in cancer and immune regulation. However, functional assays that directly measure v{beta}8-mediated activation of latent TGF-{beta} in a physiologically relevant context remain limited. Here, we report a co-culture cell-based reporter assay for quantitative measurement of v{beta}8-mediated activation of latent TGF-{beta}1. NIH/3T3 reporter cells were engineered to express a SMAD-responsive NanoLuc reporter, constitutive firefly luciferase for internal normalization, and cell-surface GARP-latent TGF-{beta}1. When co-cultured with v{beta}8-expressing LN-229 cells, reporter cells produced a robust signal that directly reflected localized latent TGF-{beta}1 activation. The assay demonstrated stable expression of the required biological components, reproducible signal-to-background performance, and sensitivity to benchmark v{beta}8-blocking antibodies. Inhibition studies showed potent dose-dependent blockade by an anti-v{beta}8 antibody. In contrast, pan-TGF-{beta} neutralizing antibody displayed markedly weaker apparent potency, suggesting that targeting localized v{beta}8-mediated activation is more effective than neutralizing released TGF-{beta} in this assay context. The assay also enabled screening and ranking of anti-v{beta}8 antibodies, identifying several high-potency clones, and detected v{beta}8-mediated activation of a non-cleavable latent TGF-{beta}1 mutant. This platform provides a sensitive, internally normalized, and scalable approach for mechanistic studies and therapeutic discovery targeting the v{beta}8-TGF-{beta} axis.

This step-by-step protocol provides a cross-species, non-surgical approach that enables prenatal gene delivery to the developing nervous system in rats and marmosets. Under transabdominal ultrasound guidance, intracerebroventricular injection of recombinant adeno-associated virus vectors into the fetal brain achieves robust and long-term transduction from prenatal stages into adulthood. This approach can be adapted to other species and target sites outside nervous system, enabling safe and selective intrauterine manipulation and the generation of diverse experimental models for basic and preclinical research. For complete details on the use and execution of this protocol, please refer to Ribeiro Gomes et al (2026)1. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/737050v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@696364org.highwire.dtl.DTLVardef@fc3c7forg.highwire.dtl.DTLVardef@1e7c7caorg.highwire.dtl.DTLVardef@1edcef0_HPS_FORMAT_FIGEXP M_FIG C_FIG Before you beginExperimental procedures during gestation allow researchers to study developmental processes, including how manipulations of the fetus and its intrauterine environment influence biological outcomes. Ultrasound imaging guidance greatly facilitates such interventions by providing safe and targeted access to fetal compartments, including for prenatal gene delivery to developing neural cell populations. Critically, delivery of recombinant adeno-associated viruses (rAAVs) into the cerebrospinal fluid (CSF) of developing animals enables widespread gene transfer across the brain. The efficiency and distribution of transduction are strongly influenced by developmental stage, making the timing of delivery an important experimental variable. In altricial species such as mice, major developmental processes, including cortical lamination and the establishment of long-range connections, begin prenatally but continue throughout early postnatal life. In primates, however, development is more advanced at birth, and many equivalent developmental events are shifted to the prenatal period. Consequently, developmental stages that can be targeted postnatally in mice require prenatal access in primates. Here, we present a step-by-step protocol for ultrasound-guided fetal intracerebroventricular viral injection (FIVI) of rAAV in marmosets (Callithrix jacchus) and rats (Rattus norvegicus). The procedure was initially developed and optimized in rats before being translated to marmosets, small New World primates that share key developmental, anatomical, and functional characteristics with humans. Together, these models illustrate the cross-species applicability of the approach, while providing gene delivery strategies for both a genetically tractable rodent model and a translationally relevant nonhuman primate. FIVI enables broad gene transfer and stable, long-term transgene expression in wild type animals, facilitating the generation of complementary quasi-transgenic models for research and translational applications from prenatal development through adulthood.

1. Video-tracking programs have now become an essential tool for researchers measuring animal behavior across biological fields. The panel of available programs is growing rapidly, providing researchers with numerous specific tools that will match their precise needs. However, their proliferation may complicate post-tracking data processing, and some programs do not even provide tools for correcting tracking errors or analysing tracking data. In the case of commercial software, the loss of access to a program due to budget limitations or researchers' mobility from one institution to another could prevent them from accessing and visualizing their tracking data. 2. There is therefore a growing need for an accessible and flexible tool to handle post-tracking processes such as the correction and analysis of tracking data obtained across different video-tracking programs. 3. We present here the latest update of the video tracking and analysis program AnimalTA. With this new release, we propose to solve the above-mentioned problems by providing the scientific community with a program that will allow for data importation from other video-tracking programs. Like in its previous versions, AnimalTA remains a free, open-source, and highly user-friendly program, ensuring that it will always be accessible without restriction. Now, with this new importation option, users who performed their tracking with other programs can benefit from AnimalTA's complete toolset of data visualization, correction, and analysis. 4. Finally, this article gives an overview of the other main improvements associated with this new release. The program is now faster in both video importation and tracking, proposes an amplified toolset for data visualisation and correction, and features new options for data analysis.

Ribonucleic acid (RNA) modifications, with over 170 identified types, play diverse roles in cellular processes. The past decade has witnessed surging demand for accurate identification and localization of RNA modifications in both endogenous and synthetic therapeutic RNAs. With accurate spectral annotation for RNA, tandem mass spectrometry (MS/MS) can meet this demand. Here we present RNabel, a user-friendly software tool for in-depth annotation of MS/MS spectra of RNA oligonucleotides. RNabel considers a full set of backbone-cleavage ions (a, b, c, d, a-B, w, x, y, z) in which the ribonucleotide unit could be A, U, C, G, Y (pseudouridine), or I (Inosine). Additionally, RNabel considers 196 modifications on the base, the phosphoribose linkage, the 5' or the 3' terminus, or detachment of a sub-nucleotide fragment as a neutral or charged group. Users can create new components if needed, including ribonucleotides, modifications, neutral or charged groups that could detach from a ribonucleotide. RNabel efficiently processes large datasets in four acceptable formats including .mgf, .raw, .txt from msConvert, and RNabel batch files. Multiple statistical metrics are provided for quality assessment of spectral annotation. To accelerate RNA modification analysis, RNabel is made freely available for Mac and Windows users at https://github.com/songge1111/RNabel/releases. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/733900v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@8ccae5org.highwire.dtl.DTLVardef@15c8cfaorg.highwire.dtl.DTLVardef@12b93a2org.highwire.dtl.DTLVardef@1e9aab9_HPS_FORMAT_FIGEXP M_FIG C_FIG

Glycans coat the surface of all cells, and every glycan is recognised by specific glycan-binding pro-teins (GBPs). There are no general tools that can accurately estimate the binding strength between glycan and GBP from the amino acid sequence of the GBP and the molecular structure of the glycan, represented as SMILES string. We describe models for predicting such binding strengths developed as a part of a Capstone Course at the University of Alberta. The models are trained on a dataset that combines BindingDB, a published database of small-molecule protein interactions, and data from glycan arrays measured by Consortium of Functional Glycomics (CFG). In this hybrid dataset of protein-ligand interactions the ligands are both glycans from CFG and small molecules from BindingDB; similarly, proteins include GBP and proteins from BindingDB. Three models are presented (i) ProMax which fuses ESM-2, MolFormer, and MolCLR features; (ii) APEX which constrains learning to a predetermined form, a physical model of binding; (iii) UltraMax adds inter-atomic distances for the ligands. To address the dataset's severe long-tail distribution, the models employ tail-aware losses for rare high-binding instances. Trained and evaluated on approximately one million protein--ligand pairs using hold-out splits for unseen molecules, the three models provide a unified framework for quantitative glycan-protein binding prediction. We observed that learning glycan-protein binding is harder than the similar task of learning small-molecule-protein interactions. Simple mirror-inversion tests led us to postulate that insufficient use of chiral features is an important source of difficulty in learning these interactions.

Targeted knock-in technologies have enabled precise insertion of reporters, affinity tags, degrons, and other functional payloads into endogenous genomic loci. Over the past decade, a diverse collection of genome engineering strategies has emerged, including approaches based on homology-directed repair (HDR), microhomology-mediated end joining (MMEJ), homology-mediated end joining (HMEJ), and related methodologies. While these advances have greatly expanded the capabilities of endogenous genome engineering, they have also increased the complexity of donor design, assembly, and validation. Here, we describe FORGE-KI (Functional Oncology Research Genetic Engineering - Knock in), a pathway-matched design workflow for endogenous knock-in engineering that aligns the assembly strategy with the underlying repair mechanism. For large-cargo insertions, we use a modular five-component framework that separates gene-specific targeting arms from reusable functional modules, allowing rapid assembly of HDR donor constructs targeting AHR, IRF1, and FOSL1 from a shared reagent collection. For MMEJ/PITCh applications, where short targeting elements permit rapid fabrication, we developed a streamlined one-step pipeline in which the entire donor and selection payload is synthesized as a single continuous fragment for direct cloning, compressing the design-to-reagent cycle time. This MMEJ workflow is paired with a dual-promoter nuclease vector (pForge-KI-MMEJ-Cas9-DualGuide) that drives the PITCh-release and locus-specific guides from distinct promoters, a design intended to reduce the repeated-promoter instability associated with some dual-guide vectors. We also established a standardized workflow for donor assembly, generation of knock-in cell populations, molecular validation, and selectable-cassette removal, and we demonstrate it by generating a functional, selection-marker-free, cytokine-inducible IRF1 HDR reporter line and an inducible IRF1 PITCh/MMEJ reporter pool with confirmed junction enrichment. In parallel, we developed forgeKI, an R package that automates C-terminal reporter knock-in design across both HDR and PITCh/MMEJ repair pathways, including guide selection, target-biology validation, targeting-arm design, domestication, donor-assembly planning, and generation of synthesis-ready constructs. Together, the reagents and software provide a practical system for endogenous knock-in engineering that supports multiple payloads, selection strategies, and repair pathways within a shared donor organization. Rather than replacing existing knock-in technologies, this framework provides a modular foundation for incorporating, extending, and automating the published knock-in methods.

To facilitate the translation of NF1 exon 17 skipping as a mutation-specific therapy for Neurofibromatosis type 1 into in vivo testing, we have continued to develop more efficient antisense oligonucleotides (ASOs), humanized mouse models, and explored multiple delivery platforms including an adeno-associated virus (AAV)-U7-SnRNA vector approach. We evaluated both biodistribution and exon skipping efficacy of a U7-SnRNA targeting NF1 exon 17 with an SFFV-driven cassette containing T2A-linked Luciferase (Luc) and eGFP packaged in AAV-9, AAV-F and AAV-B1 capsids. We show that AAV-F is superior to AAV-9 and AAV-B1 for mouse brain delivery based on DNA transduction, GFP expression, and luciferase activity, but AAV-B1 delivers 2-4 fold more to sciatic nerve (SCN). In terms of exon skipping, AAV-F appears to induce the most skipping in liver and optic nerve (ON), while AAV-B1 mediates highest skipping in the liver, SCN, and ON. The identification of AAV serotypes that allow efficient transduction and delivery of transgenes to the mouse CNS and PNS is impactful for preclinical research in murine models of other diseases. Furthermore, this is both the first report of NF1 exon skipping efficacy in vivo and the first successful application of an U7-SnRNA for the restoration of functional neurofibromin for NF1.

DNA data storage allows sequences to be defined without biological constraints, yet readout workflows still depend on generic end-repair/dA-tailing chemistry. We developed NinjaSeq, a type IIS restriction endonuclease library-preparation strategy that incorporates recognition sites into primer flanks, enabling digestion to generate adapter-compatible overhangs and eliminating the need for conventional end preparation. By combining this chemistry with constrained coding that excludes internal recognition motifs, NinjaSeq produced sequencing quality and decoding performance consistent with standard protocols while reducing reagent burden and simplifying processing, including compatibility with one-pot restriction-ligation. The same sequence-directed design also enables physical random access during library preparation: targeting file-specific flanking sites enriched a desired file from a mixed pool by about sixteen-fold in a proof-of-concept experiment. These results position NinjaSeq as a practical ONT readout approach for DNA data storage. HIGHLIGHTSO_LINinjaSeq replaces end-repair/dA-tailing with REases for nanopore sequencing C_LIO_LIConstrained encoding excludes recognition motifs to protect payloads from cleavage C_LIO_LINinjaSeq achieves decoding accuracy comparable to standard library preparation C_LIO_LIDesigning file-specific RRS enables random access during library preparation C_LI

Here we introduce OpenEvo, a fully open-source, low-cost turbidostat platform for automated continuous culture and directed evolution experiments. Existing tools are expensive, complex, or lack open-source hardware; OpenEvo addresses this gap. OpenEvo is a complete, fully automated evolution platform with detailed, illustrated construction instructions for beginners, open-source software and firmware, and a single device priced around $300. An optional PC-based version offers enhanced functionality, including remote access, programmable evolution cycles, programmable LED stimulation, and a data visualization tool. OpenEvo can cycle through three types of media for positive, negative, and neutral selection conditions, supporting a wide range of experimental designs. We validate the use of OpenEvo by evolving H. volcanii to grow from 15% to 12% salt over ~150 cycles, ~1,000 hours. Evolved cells grew 36% faster than wild-type at 12% salt. Whole-genome sequencing of adapted cells found SNPs and large deletions. We also demonstrate positive and negative selection using the OpenEvo LEDs to drive optogenetics via a Phytochrome B-based optogenetic tool, with light as the selection stimulus during over 4000 hours of growth. OpenEvo lowers the technical and cost barriers for continuous evolution experiments, serves as a teaching tool, and is designed to grow an open community of users who share modifications.

Imaging Mass Cytometry (IMC) combines metal-tagged antibody labelling with laser ablation mass spectrometry to generate highly multiplexed spatial images of tissue sections. However, the area that can be acquired within a single region of interest (ROI) is limited by hardware and software constraints, requiring large tissues to be imaged as multiple tiled ROIs. Reconstructing these ROIs into whole-slide images requires additional processing, while the proprietary .mcd file format can hinder integration with standard bioimage analysis workflows. Here, we present MCD Stitcher, an open-source Python package for converting .mcd files into OME-TIFF images with automated whole-slide stitching. The tool supports rectangular and polygonal ROIs, accommodates variable pixel sizes between ROIs, and uses memory-aware chunked reading during data ingestion to process large datasets on standard workstations. The generated OME-TIFF outputs preserve spatial, channel, and acquisition metadata for downstream analysis in tools such as QuPath, napari, and ImageJ/Fiji. MCD Stitcher provides a reproducible workflow for converting raw IMC data into interoperable image formats, enabling whole-slide spatial analysis without reliance on vendor-specific software.

Survival analysis is a workhorse assay in Drosophila research to evaluate somatic fitness. It is indispensable in the study of ageing and insightful in immunity, metabolism, radiobiology, toxicology, ecology, and others. While conceptually simple, lifespan measurement is labour-intensive because it requires the continuous manual maintenance of large experimental cohorts. Here, we describe Drosben, an approach that combines a 3D-printed device to transfer flies from several vials simultaneously, a paper system for quick data recording and accompanying software that automatically digitalises life tables for analysis. We show that using Drosben reduces the time investment to perform lifespan assays by ~85%, with improved speed regardless of experience handling Drosophila vials. Using Drosben, we address the effects on longevity of chronic feeding of indole-acetic acid (IAA), naphthalene-acetic acid (NAA) and trimethoprim (TMP) -- compounds used to control heterologous targeted protein degradation systems. We find that IAA and NAA have noticeable deleterious effects while TMP has a small protective effect specifically in females. We further show that strong static magnetic fields do not affect Drosophila lifespan. Our work suggests that Drosben can cheaply accelerate research where lifespan is used as a life history trait.

Zebrafish combines the power of genetics and unparalleled in vivo imaging for investigating the dynamics of vertebrate hematopoietic development. Across species, the transcription factor Runx1 is essential for definitive hematopoiesis. We generated a zebrafish runx1-2A-creERT2 CRISPR knock-in for tamoxifen-regulated Cre recombinase Runx1 lineage tracing and characterized its activity using the ubi:Switch recombinase-dependent fluorescence reporter, microscopic live imaging and flow cytometry. Tamoxifen treatment beginning at gastrula stage labeled all expected Runx1 lineages in the early embryo, including neuroectodermal olfactory placode and Rohan-Beard neurons, primitive hematopoietic blood cells, and nascent hematopoietic stem and progenitor cells (HSPCs) in the dorsal aorta. Runx1 HSPCs colonized the larval caudal hematopoietic tissue and thymus from three to five days of development. Timed tamoxifen induction of Cre activity allowed separation of Runx1 primitive hematopoiesis from definitive HSPC emergence and larval stem cell niche colonization. Flow cytometry of kidney marrow and peripheral blood from adults treated with tamoxifen at gastrula stage revealed Runx1 embryonic hematopoietic cells contributed to adult hematopoietic precursors, myeloid, lymphoid, and peripheral blood lineages. Labeling of all blood lineages was also effective by tamoxifen treatment of 5-month-old adults. The zebrafish runx1-2A-creERT2 line provides a powerful tool for precise spatial and temporal analysis of Runx1 progenitor mechanisms in developmental and adult hematopoiesis. Key PointsO_LIzebrafish endogenous runx1-2A-creERT2 provides inducible Cre recombinase genetic analysis in all runx1 neuromesodermal and blood lineages C_LIO_LIzebrafish runx1-2A-creERT2 line enables in vivo spatial and temporal analysis of embryonic and adult hematopoiesis C_LI

Whole-genome sequencing identifies focal DNA amplifications with base-pair resolution but cannot determine whether amplified sequences reside on extrachromosomal DNA (ecDNA, also known as double minutes) or within chromosomally integrated homogeneously staining regions (HSRs). DNA fluorescence in situ hybridization (DNA-FISH) metaphase spreads remain the gold standard for distinguishing these amplification states at single-cell resolution. Here, we present a detailed protocol for DNA-FISH metaphase spreads using human cancer cell lines, encompassing cell culture, metaphase arrest, hypotonic treatment, fixation, chromosome spreading, fluorescent probe hybridization, and fluorescence imaging. The protocol incorporates intermediate quality-control steps to verify successful chromosome dispersion and optimize metaphase spread quality, making the workflow accessible to laboratories without specialized cytogenetics expertise. Results demonstrate clear visualization of ecDNA and HSR amplification states using locus-specific probes and illustrate common technical artifacts that can affect interpretation. This protocol provides a robust and reproducible approach for studying the structural organization of oncogene amplification in cancer cells.

Precise regulation of Drosophila Myc is essential for growth and homeostasis, yet regulation of its transcriptional control remains incompletely understood. We investigated the Myc cis-regulatory landscape using in vivo reporter assays, EMSA, and LC-MS/MS-based identification of DNA-associated proteins. By truncating Myc cis-regulatory modules (CRMs), we delineated the activity of conserved non-coding elements across adult female tissues and larval stages. Specific DNA-protein interactions were confirmed by EMSA using nuclear embryonic extracts. We developed a Solid Surface Magnetic Enrichment protocol (SSMEP) to pull down DNA-protein complexes formed on Myc cis-elements. Affinity purification followed by LC-MS/MS enabled the identification of candidate transcriptional regulators associated with Myc-CRMs. This integrative approach provides new insights into promoter structure and trans-regulatory architecture of Myc and their roles in developmental gene expression programs. Our study identifies a distal enhancer required for larval and pupal patterning, with activation dependent on specific spacing relative to the TATA-box core promoter, a strong enhancer cluster within 5'-UTR cis-elements active in ovaries and embryos, and a DPE-core promoter requiring nearby enhancer action. The DPE-linked enhancer can function with both Inr-DPE and TATA-box promoters.The identified Myc-CRMs interact with conserved signaling pathways to tightly control Myc during development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/736081v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@fa98a9org.highwire.dtl.DTLVardef@3c82d3org.highwire.dtl.DTLVardef@b1025corg.highwire.dtl.DTLVardef@111e972_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMyc Oocyte Element: an eRNA-producing enhancer in ovaries and early embryos C_LIO_LIDPE promoter synergizes with nearby enhancer to drive Drosophila Myc transcription C_LIO_LILate enhancer licenses TATA promoter for larval tissue-specific Myc transcription C_LIO_LIPromoter-enhancer dynamics differentially drive Myc during development C_LIO_LISSMEP protocol helps purify and enrich low-abundance DNA-protein complexes C_LI

BackgroundScratch assays are widely used to study wound closure in vitro, but quantitative image analysis remains constrained by manual variability, proprietary workflows, and tools requiring programming expertise. We developed InVitroGap, a Python-based application with a browser-accessible interface for automated quantification of scratch assay closure from sequential microscopy images. MethodsRCC-ER and Renca cells were seeded in 96-well ImageLock plates and scratched using a WoundMaker device for uniform linear wounds or a 200 {micro}L pipette tip for crisscross wounds. Phase-contrast time-lapse images acquired at 0, 24, and 48 h with an IncuCyte SX5 system were independently analyzed using IncuCyte 2023A Rev2 and InVitroGap. The InVitroGap pipeline combines Gaussian smoothing, gradient-based texture mapping, adaptive percentile thresholding, and morphological post-processing to quantify wound confluence and relative wound density (RWD). Agreement was evaluated using paired comparisons, Pearson and Spearman correlations, Bland-Altman analysis, and mean absolute error (MAE). ResultsInVitroGap measurements closely tracked IncuCyte outputs across both cell lines, with no significant between-method differences (p > 0.05), strong pooled correlations (R{superscript 2} = 0.964 for RWD; R{superscript 2} = 0.983 for wound confluence), and small mean biases (absolute bias [≤] 1.64%). The tool successfully processed crisscross wounds from brightfield image series, and a complete four-timepoint series was analyzed in approximately 10 seconds, with robust performance across distinct cell morphologies and wound geometries. ConclusionsInVitroGap provides a transparent, computationally efficient, and platform-independent alternative for scratch assay analysis, delivering performance comparable to commercial systems while remaining freely accessible at https://invitrogap.vercel.app/. HighlightsO_LIOpen-source Python tool for automated, platform-independent in vitro scratch assay analysis C_LIO_LITexture-based adaptive pipelines enable robust wound segmentation across cell types C_LIO_LIQuantifies wound confluence and relative wound density from time-lapse images C_LIO_LIStrong agreement with IncuCyte measurements in the tested datasets C_LI

Glycopeptide enrichment remains a cornerstone in glycoproteomics, but bias and reproducibility issues continue to hinder biological insight and clinical translation. Using curated glycoproteomics datasets and machine learning, we trained a glycopeptide classifier to promptly recognize N-glycopeptide precursor ions in peptide mixtures through mass defect signatures. Integration of the classifier into a data-dependent acquisition framework facilitated efficient and unbiased real-time prediction of N-glycopeptides directly from serum opening avenues for enrichment-free glycoproteomics.

Targeted long read sequencing (LRS) of native genomic DNA (gDNA) using Oxford Nanopore Technologies (ONT) is an economically and computationally accessible method for sequencing selected genomic regions without the limitations associated with amplification-based approaches. At present, efficiency, multiplexing, and scalability remain key challenges for existing targeted LRS. We have developed Cas12a-Targeted Multiplexed Nanopore Sequencing (CTM-nSeq), which combines Cas12a-targeting, DNA fragment enrichment, and optimized adapter ligation using T7 DNA ligase. Unlike previously established protocols, CTM-nSeq is compatible with the latest ONT flow cell chemistry. Performing CTM-nSeq on a single sample with an R10.4 MinION flow cell routinely yields hundreds of on-target reads. Furthermore, CTM-nSeq enables targeting of multiple loci and is the first targeted ONT sequencing method, allowing reliable, barcode-assisted multiplexing. CTM-nSeq is an efficient and accessible method for sequencing native gDNA and analysing DNA methylation, repeat expansions, and sequence integrity. As such, CTM-nSeq has a wide range of analytical and diagnostic applications.

The role of antigen-specific T cells responding to antigen is a topic of intense studies, and critical for mechanistic insight of diseases and development of therapeutic strategies. Methods for broad-scale detection of antigen-specific CD4 T cells are lacking, while such methods have demonstrated great value in exploring CD8 T cell response in health and disease. Furthermore, major histocompatibility complex II (MHCII) assays are technically challenging due to high HLA diversity, lower binding affinities, low frequencies of ex vivo antigen-specific CD4 T cells and several bottlenecks in production and peptide exchange of MHCII monomers. Here we use peptide-loaded MHCII (pMHCII) proteins multimerized on a barcode- and fluorophore-labelled dextran backbone to provide a method for the detection of peptide-specific CD4 T cells by using a large display of MHCII-associated peptides. We have established a protocol for MHCII production and peptide-exchange suitable for the generation of large libraries of peptide-MHCII complexes. We validate the use of such pMHCII complexes in the form of barcode-labelled MHCII multimers to detect antigen-specific CD4 T cells. We demonstrate that we can identify antigen specific CD4 T cells, using these DNA barcoded peptide-MHCII multimer. The multimer bound CD4 T cells were selected based on the fluorochrome signal, and the co-attached DNA barcodes were hereafter amplified and used to identify the peptide-MHCII response/binding. In cases where the peptide-specific CD4 T cells frequencies are very low, we expanded the cell population with peptide-pools and in the presence of IL2. The given CD4 T cell populations hereby reach a cell number allowing for the DNA-barcoded pMHCII multimers to detect responses otherwise missed out. Applying this technology, we utilized a panel of 150 peptides derived from human cytomegalo virus (CMV), Epstein barr virus (EBV), Influenza (Flu), SARS CoV 2 and SARS CoV1, Hepatitis B virus (HBV), and Hepatitis C virus (HCV) loaded onto HLA-DRB1*01:01 and DRB1*04:01 to screen peripheral blood mononuclear cells (PBMC). We assessed ex vivo responses in 16 participants with HCV infection, and successfully detected naturally occurring viral-specific CD4 T cells at frequencies as low as 0.004% of total CD4 T cells. The low-frequency responses, identified via the barcode screen, were rigorously validated using individual fluorophore-labelled tetramer staining after a peptide-driven expansion in 15 participants. Furthermore, we assessed the recognition of novel HCV epitopes in 11 additional participants. Through this, we identified a total of 12 distinct HCV epitopes, including 9 that have not been previously utilized in assays to detect CD4 T cells. Overall, this barcoded-multimer platform provides a powerful tool for the large-scale discovery of class II epitopes and the broad profiling of CD4 T cell specificities. This method will allow for in-depth analyses of immune interactions, provide a better understanding of the antigen-driven associations between CD4 and CD8 T cell responses, and help dissect the complexities of CD4 T cell protection in HCV infection.