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| Item Type: | Preprint |
|---|---|
| Title: | Cryo-mtscATAC-seq for single-cell mitochondrial DNA genotyping and clonal tracing in archived human tissues |
| Creators Name: | Salla, Maren, Obermayer, Benedikt, Cotta, Marie, Friebel, Ekaterina, Campo-Garcia, Juliana, Charalambous, Georgia, Bueno, Roemel Jeusep, Lieu, Dustin, Dabek, Patryk, Helmuth, Ashley, Tellides, George, Assi, Roland, Bankov, Katrin, Lodrini, Marco, Deubzer, Hedwig, Beule, Dieter, Chung, Hattie, Radbruch, Helena, Capper, David, Heppner, Frank, Starossom, Sarah C., Lareau, Caleb A., Liu, Ilon and Ludwig, Leif S. |
| Abstract: | High-throughput clonal tracing of primary human samples relies on naturally occurring barcodes, such as somatic mitochondrial DNA (mtDNA) mutations detected via single-cell ATAC-seq (mtscATAC-seq). Fresh-frozen clinical specimens preserve tissue architecture but compromise cell integrity, thereby precluding their use in multiomic approaches such as mitochondrial genotyping at single-cell resolution. Here, we introduce Cryo-mtscATAC-seq, a broadly applicable method for diverse pathophysiological contexts to isolate nuclei with their associated mitochondria (“CryoCells”) from frozen samples for high-throughput clonal analysis. We applied Cryo-mtscATAC-seq to the neurodegenerated human brain, glioblastoma (GBM), pediatric neuroblastoma, and human aorta, and implemented mitobender, a computational tool to reduce ambient mtDNA in single-cell assays. Our approach revealed regional clonal gliogenesis and microglial expansions in amyotrophic lateral sclerosis (ALS), persistence of oligodendrocyte progenitor cell (OPC)-like clones in GBM recurrence, mtDNA depth heterogeneity after neuroblastoma chemotherapy, and oligoclonal proliferation of smooth muscle cells in human aorta. In conclusion, Cryo-mtscATAC-seq broadly extends mtDNA genotyping to archival frozen specimens across tissue types, opening new avenues for investigation of cell stateinformed clonality in human health and disease. |
| Keywords: | Animals, Mice |
| Source: | bioRxiv |
| Publisher: | Cold Spring Harbor Laboratory Press |
| Article Number: | 2025.09.17.675534 |
| Date: | 20 September 2025 |
| Official Publication: | https://doi.org/10.1101/2025.09.17.675534 |
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