The core idea is elegantly simple. A fluorescent tracer dye called Fast Blue is applied to the organ of interest. Neurons whose axons terminate in that organ take up the dye at their nerve endings and transport it backward along their axons to their cell bodies, a process known as retrograde axonal transport. A few days later, the relevant ganglia are dissected, dissociated into single cells, and passed through a fluorescence-activated cell sorter. Only the fluorescently labeled neurons, those with a proven physical connection to the target tissue, are retained. Each captured cell is then subjected to single-cell RNA sequencing, generating a full transcriptome for every traced neuron.

The crucial advance over existing approaches lies in selectivity. Sequencing a whole ganglion without labeling captures everything: sensory neurons serving skin and muscle, autonomic neurons innervating unrelated organs, satellite glial cells and other stromal populations. That background can completely swamp the small subset of neurons connected to the tissue a researcher actually cares about. Bulk sequencing is even blunter, averaging signals across thousands of cells and erasing neuronal subtype identity altogether. Trace-n-Seq eliminates this noise by design, allowing researchers to characterize organ-specific neurons at single-cell resolution and to detect even rare tissue-innervating subsets that would otherwise be invisible.

The complete workflow, published in Nature Protocols by Vera Thiel, Manuel Mastel, Simon Renders and colleagues in the laboratories of Martin Sprick and Andreas Trumpp, can be completed in about two weeks. It encompasses tracer application, ganglion dissection, enzymatic tissue digestion, careful neuronal validation, fluorescence-based sorting, SMART-seq2 library preparation and a dedicated bioinformatic pipeline. The published protocol walks readers through each stage with schematic illustrations, dissection guidance, quality-control checkpoints and computational scripts. The authors note that the method suits researchers experienced in molecular biology, neuronal tissue handling, sequencing techniques and bioinformatics, and they have deposited their complete Seurat-based analysis workflow, quality-control scripts and annotation code openly on GitHub. The underlying sequencing data are accessible through the ArrayExpress repository under accession E-MTAB-12940.

Why does this matter biologically? Neuronal innervation is now recognized as a master regulator of tissue function well beyond classical sensation and motor control. Nerves shape organ function during homeostasis and regeneration, and they are increasingly implicated in pathological settings including inflammation, autoimmune disease, fibrosis and cancer. The emerging field of cancer neuroscience, highlighted in the protocol’s own key reference by Monje and Winkler, argues that tumors are not just passive targets of nerve growth but active participants in a dialogue with the nervous system. Tumor-associated nerves can be reprogrammed by the tumor microenvironment, and in some contexts these remodelled neurons appear to support malignancy itself.

Trace-n-Seq was developed and validated in exactly such a context. The protocol builds on the team’s earlier research published in Nature in 2025, in which they characterized single neurons reprogrammed by pancreatic cancer. Pancreatic ductal adenocarcinoma is one of the most densely innervated human tumors, and the earlier study showed that individual neurons connected to the tumor acquire distinct transcriptional states compared with neurons innervating healthy tissue. By tracing neurons from the tumor back to their ganglia and sequencing them one by one, the researchers could separate genuine disease-associated neuronal states from the bulk signature of the ganglion. The new protocol packages that experimental logic into a step-by-step recipe that any qualified laboratory can follow.

The versatility of the method is a major selling point. Because it depends only on functional connectivity, Trace-n-Seq is in principle applicable to any healthy, inflamed or diseased tissue innervated by the peripheral nervous system. Researchers can compare traced neurons from a healthy pancreas with those from a pancreatitis model or a tumor-bearing organ, directly quantifying how disease remodels neuronal subtype composition and gene expression. They can capture rare tissue-specific neuronal subsets, distinguish disease-associated states from background ganglia signatures, and follow neuronal plasticity across physiological and pathological conditions at single-cell resolution. This functional, connectivity-based profiling contrasts with purely anatomical or genetic labeling strategies and provides an orthogonal view of peripheral neuron diversity.

The technical details matter for anyone hoping to reproduce the approach. Fast Blue is a retrograde tracer well established in neuroanatomy, valued for its intense fluorescence and compatibility with downstream flow cytometry. After allowing sufficient time for axonal transport, ganglia are harvested and must be gently digested to yield viable, intact neurons, a step the authors emphasize with dedicated validation procedures to confirm that sorted cells are indeed neurons rather than debris or contaminating glia. Fluorescence-activated cell sorting then enriches the labeled population before library construction. The bioinformatic workflow includes quality control, filtering and annotation steps tailored to traced neurons, including reference-based neuronal annotation, so that researchers can confidently classify traced cells into known sensory and autonomic subtypes and discover novel disease-associated clusters.

The publication arrives at a moment of rapid growth for the neuro-immune and neuro-oncology fields. Other studies have revealed, for example, that lymph nodes are innervated by a unique population of sensory neurons with immunomodulatory potential, and barcoded viral tracing approaches have begun to map single-cell interactions during central nervous system inflammation. Large-scale atlases from single-cell sequencing efforts have catalogued the molecular architecture of the mouse nervous system, classifying sensory and visceral motor neuron types in unprecedented detail. What these atlases lack, however, is information about where each neuron actually projects. Trace-n-Seq adds that anatomical dimension, linking molecular identity to functional tissue connectivity in a single experiment.

For clinical translation, the implications are considerable. If neurons innervating tumors can be identified and molecularly profiled, they become potential therapeutic targets or biomarkers. Clinically actionable strategies for studying neural influences in cancer have been a subject of active discussion, and tools like Trace-n-Seq provide the granular data needed to move from observation to intervention. Understanding which neuronal subprograms a tumor co-opts, and how inflammatory or fibrotic diseases rewire peripheral innervation, could eventually inform drugs that interrupt harmful neuro-tissue signaling while sparing essential nerve function. The open release of the protocol, the analysis code and the source datasets lowers the barrier for laboratories worldwide to adopt the technique, and the authors declare no competing interests. As neuroscience and organ biology continue to converge, methods that reveal which neurons talk to which tissues, and what they say at the transcriptomic level, are likely to become standard equipment in the effort to understand how the nervous system shapes health and disease.

Subject of Research: A single-cell sequencing protocol combining retrograde fluorescent tracing and cell sorting to molecularly profile peripheral neurons innervating specific tissues.

Article Title: Trace-n-Seq combines retrograde fluorescent tracing with sorting and single-cell sequencing of innervating peripheral neurons

Article References: Thiel, V., Mastel, M., Renders, S., Panten, J., Bauer, K., Jackstadt, R., Sprick, M. R., & Trumpp, A. (2026). Trace-n-Seq combines retrograde fluorescent tracing with sorting and single-cell sequencing of innervating peripheral neurons. Nature Protocols. https://doi.org/10.1038/s41596-026-01416-z

Image Credits: AI Generated

DOI: 10.1038/s41596-026-01416-z

Keywords: Trace-n-Seq, retrograde tracing, single-cell RNA sequencing, peripheral neurons, fluorescence-activated cell sorting, ganglia, cancer neuroscience, neuronal innervation, Fast Blue, pancreatic cancer, Nature Protocols, neuron-tissue interactions