The challenge that the method, called VINE-seq, was designed to solve is fundamentally mechanical. Most single-nucleus sequencing workflows begin by gently breaking brain tissue apart and releasing nuclei from individual cells. That approach works reasonably well for neurons and glia, but vascular cells are a different story. They are encased within a tough basement membrane, a dense meshwork of proteins that resists standard dissociation methods. As a result, when researchers homogenize brain tissue for single-nucleus profiling, vascular cells are systematically lost, depleted, or damaged. The consequence has been a major blind spot in the single-cell atlases of the human brain that now underpin much of modern neuroscience: the very cells that form the blood-brain barrier and regulate cerebral blood flow are dramatically underrepresented in the datasets used to study health and disease.

VINE-seq, short for vessel isolation and nucleus extraction for sequencing, tackles the problem head-on by isolating the vessels themselves before any attempt is made to extract their genetic material. The workflow begins with fresh or frozen brain tissue, either human or mouse, which is homogenized under conditions that preserve vascular integrity. The homogenate is then subjected to dextran-based density-gradient centrifugation, a technique that exploits differences in buoyant density to separate intact vessels from the surrounding myelin and the parenchymal fraction containing neurons and glia. The vessels recovered by this step are predominantly capillaries and small arterioles and venules, roughly 100 micrometers in diameter or smaller, which represent the functional workhorses of the cerebral circulation.

Once the vascular pellet has been collected, the protocol calls for rigorous washing of the isolated vessels over a cell strainer. This deceptively simple step removes trapped contaminants, including parenchymal cells and debris that can cling to the outside of vessel fragments and muddy downstream molecular profiles. The critical innovation, however, comes in the third stage: the optimized extraction of nuclei from the purified vessels using enzymatic digestion, specifically with collagenase III. The authors report that the precise conditions of this digestion step were tuned to liberate intact nuclei from endothelial, mural, and perivascular cells without compromising their quality, a balance that had defeated earlier attempts to profile vascular cells from frozen tissue.

After extraction, the protocol employs fluorescence-activated cell sorting, or FACS, to ensure that only high-purity nuclei proceed to sequencing. This sorting step is what makes the method compatible with the droplet-based platforms that dominate the field, including the widely used 10x Genomics single cell 3′ gene expression assay and the 10x multiome platform, which captures both gene expression and chromatin accessibility from the same nucleus. In parallel with the vascular nuclei, the protocol recovers parenchymal nuclei from the density gradient, meaning that a single dissection can yield both a vascular and a non-vascular view of the same tissue sample. Both fractions are purified by FACS, ensuring compatibility with single-nucleus RNA sequencing, single-nucleus ATAC sequencing, and combined multiomic workflows.

The practical demands of the protocol are modest by the standards of modern genomics. The authors estimate that the full workflow, from homogenized tissue to sorted nuclei ready for sequencing, requires approximately four to five hours to complete. Researchers with training in single-cell techniques and flow cytometry should be able to carry it out, which lowers the barrier to entry considerably compared with bespoke methods that demand specialized instrumentation or rare expertise. The protocol also accommodates both fresh and frozen tissue, a crucial feature for human research, where most brain samples arrive from brain banks as frozen material. Detailed guidance is provided for interpreting quality-control checkpoints along the way, including the use of Agilent BioAnalyzer traces during downstream library preparation, so that laboratories can diagnose and correct problems before committing precious samples to sequencing.

The significance of the method is best understood through the discoveries it has already enabled. VINE-seq formed the technical backbone of a human brain vascular atlas published in Nature in 2022, which revealed diverse mediators of Alzheimer’s disease risk expressed in specific vascular cell populations. Its multiome successor, MultiVINE-seq, extended the approach to paired measurements of gene expression and chromatin accessibility, and underpinned a 2025 study in Neuron demonstrating that human brain vascular multi-omics can elucidate disease-risk associations that transcriptomics alone cannot resolve. By publishing the complete protocol, the authors are effectively handing the wider research community the keys to a molecular map of the cerebrovasculature that was previously accessible only to a handful of specialist laboratories.

The broader implications reach into nearly every corner of neuroscience and neurology. The blood-brain barrier, formed by tightly joined endothelial cells and supported by pericytes and astrocyte endfeet, controls what enters the brain from the bloodstream and is implicated in systemic infection, inflammation, and the failure of countless drug candidates to reach their targets. Perivascular macrophages, which the protocol captures alongside endothelial and mural cells, are increasingly recognized as immunological sentinels with roles in neurodegeneration. Recent work has linked glycocalyx dysregulation to blood-brain barrier failure in aging, identified angiopoietin signaling as a central axis of amyloid-driven vascular dysfunction, and shown that depletion of the RNA-binding protein TDP-43 in endothelial cells disrupts core barrier pathways in neurodegeneration. Each of these lines of inquiry depends on the ability to molecularly profile vascular cells from real human tissue, precisely what VINE-seq and MultiVINE-seq make routine.

The authors have also made the underlying data openly available, with raw sequencing data for VINE-seq deposited in the NCBI Gene Expression Omnibus under accession GSE163577 and MultiVINE-seq data available in the Sequence Read Archive under BioProject PRJNA1182356. For a field that has spent more than half a century developing methods to isolate brain microvessels, dating back to pioneering capillary isolation work in the 1970s, the arrival of a standardized, hours-long workflow that feeds directly into single-nucleus and multiome sequencing marks a genuine inflection point. As laboratories around the world adopt the protocol, the vascular blind spot in brain atlases is likely to shrink rapidly, and with it, the gaps in our understanding of how the brain’s plumbing shapes neurological health, aging, and disease.

Subject of Research: Single-nucleus and multiome sequencing protocols for profiling cells of the brain vasculature

Article Title: VINE-seq and MultiVINE-seq for single-nucleus and multiome profiling of the brain vasculature

Article References: Oberhauser, J., Ding, B., Reid, M. M., Xie, W. H., & Yang, A. C. (2026). VINE-seq and MultiVINE-seq for single-nucleus and multiome profiling of the brain vasculature. Nature Protocols. https://doi.org/10.1038/s41596-026-01434-x

Image Credits: AI Generated

DOI: 10.1038/s41596-026-01434-x

Keywords: VINE-seq, MultiVINE-seq, brain vasculature, single-nucleus sequencing, blood-brain barrier, endothelial cells, pericytes, perivascular macrophages, FACS, Alzheimer's disease, multiome profiling, Nature Protocols

The ISSAAC-seq protocol commences with the dual tagging of active chromatin regions, utilizing Tn5 transposase, which has become a cornerstone technique due to its efficiency and reliability. This initial phase of the workflow is crucial as it identifies regions of the genome that exhibit chromatin accessibility—an indicator of which genes may be transcriptionally active and poised for expression. By mapping these accessible regions, scientists can begin to unravel the regulatory mechanisms that dictate gene expression for specific cell types.

An essential component of the ISSAAC-seq methodology involves the generation of RNA-DNA hybrids. This is accomplished through reverse transcription, which allows the conversion of RNA into complementary DNA (cDNA). This hybridization step is a pivotal advancement, as it effectively bridges the gap between chromatin accessibility and gene expression profiling, allowing researchers to simultaneously assess both molecular layers from the same nucleus.

Following this initial tagging and hybridization, researchers have a range of options available for isolating single nuclei. The flexibility of ISSAAC-seq is evident in its compatibility with multiple single-nucleus isolation strategies. Both plate-based and droplet-based barcoding approaches can be utilized, depending on the specific needs and objectives of the study. This adaptability enhances the protocol’s versatility, making it suitable for a wide array of applications across different biological contexts.

Another remarkable feature of ISSAAC-seq is its scalability. The workflow is designed to accommodate a flexible throughput, enabling researchers to analyze anywhere from hundreds to tens of thousands of individual nuclei in a single run. This modularity is particularly advantageous for large-scale studies aimed at elucidating the heterogeneity found within complex tissue samples. By enabling high-throughput analysis, ISSAAC-seq opens new avenues for large-scale genomic studies that require robust data sets.

Notably, the robustness and sensitivity of the ISSAAC-seq methodology ensure that the resultant data generated is of high quality for both chromatin accessibility and gene expression. In the global context of genomic research, high-quality data is fundamental for drawing reliable conclusions and advancing our understanding of cellular behaviors and interactions. The coherent nature of the data produced through this dual-modality approach enhances the interpretative power of subsequent analyses.

The entire ISSAAC-seq workflow is impressively streamlined, allowing it to be completed within just one to two days. This rapid turnaround time is a significant advantage, especially for laboratories that operate in fast-paced research environments. By reducing the time required for sample preparation and data collection, researchers can focus on the downstream analysis and interpretation of their results, ultimately accelerating the pace of discovery in molecular biology.

As the scientific community continues to appreciate the importance of heterogeneity in cell populations—particularly within the context of development, disease, and therapeutic response—the ability to simultaneously profile chromatin and gene expression is increasingly invaluable. The insights gleaned from ISSAAC-seq will undoubtedly inform researchers about the cell type-specific regulatory mechanisms that govern gene expression, highlighting potential targets for therapeutic interventions.

Moreover, the potential applications of ISSAAC-seq extend far beyond basic research. Understanding chromatin accessibility and gene expression dynamics at the single-cell level carries implications for fields such as cancer research, where the heterogeneity of tumor cells can significantly impact treatment outcomes. The ability to profile both aspects concurrently may unveil crucial insights into how tumors adapt and respond to therapy, paving the way for more personalized treatment strategies.

In addition to its application in cancer research, ISSAAC-seq can be harnessed to investigate various other biological questions, including developmental biology, neurobiology, and immunology. As scientists strive to delineate the complex regulatory networks that control cellular function and fate, the dual profiling afforded by ISSAAC-seq provides a powerful tool for understanding the multifaceted nature of cellular regulation.

Looking ahead, researchers anticipate that the widespread adoption of ISSAAC-seq will catalyze a paradigm shift in how cellular biology is approached. The integration of chromatin accessibility and gene expression data at the single-nucleus level is set to become a foundational component of cellular profiling, enabling a deeper understanding of the molecular underpinnings of diverse biological phenomena.

In conclusion, ISSAAC-seq represents a significant step forward in the quest to understand cellular complexity. By allowing researchers to assess chromatin dynamics and gene expression in tandem, this innovative protocol promises to enhance our understanding of the molecular mechanisms that drive cell fate decisions across a range of biological contexts. As the field progresses, it is expected that ISSAAC-seq will continue to yield crucial insights, informing future research directions and therapeutic innovations.

Subject of Research: Multimodal profiling of chromatin accessibility and gene expression.

Article Title: Single-nucleus chromatin accessibility and gene expression co-profiling by ISSAAC-seq.

Article References:

Xu, W., Hu, Y., Zhang, Y. et al. Single-nucleus chromatin accessibility and gene expression co-profiling by ISSAAC-seq.
Nat Protoc (2026). https://doi.org/10.1038/s41596-025-01304-y

Image Credits: AI Generated

DOI: https://doi.org/10.1038/s41596-025-01304-y

Keywords: Chromatin accessibility, Gene expression, Single-cell analysis, ISSAAC-seq, Molecular profiling, Cellular heterogeneity.