In a groundbreaking advancement within the realm of cellular biology, researchers have developed a novel technique called ISSAAC-seq, which allows for the simultaneous analysis of chromatin accessibility alongside gene expression at the single-nucleus level. This innovative method aims to provide deeper insights into the intricate cellular heterogeneity that exists within tissues. Traditionally, researchers have relied on single-modality profiling approaches, which often fall short of capturing the full spectrum of molecular interactions occurring within a single cell. ISSAAC-seq addresses this limitation by enabling a more comprehensive multidimensional examination of cellular characteristics.
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.
