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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: |
Salla, Maren  ORCID: https://orcid.org/0009-0003-8453-2935, Obermayer, Benedikt ORCID: https://orcid.org/0000-0002-9116-630X, Cotta, Marie ORCID: https://orcid.org/0000-0002-2589-3189, Friebel, Ekaterina ORCID: https://orcid.org/0000-0003-1419-2376, Campo-Garcia, Juliana, Charalambous, Georgia, Bueno, Roemel Jeusep, Lieu, Dustin, Dabek, Patryk, Helmuth, Ashley, Tellides, George, Assi, Roland, Bankov, Katrin, Lodrini, Marco, Deubzer, Hedwig ORCID: https://orcid.org/0000-0002-6115-4893, Beule, Dieter ORCID: https://orcid.org/0000-0002-3284-0632, Chung, Hattie ORCID: https://orcid.org/0000-0001-8417-5606, Radbruch, Helena ORCID: https://orcid.org/0000-0001-6941-3397, Capper, David, Heppner, Frank ORCID: https://orcid.org/0000-0001-9816-8917, Starossom, Sarah C. ORCID: https://orcid.org/0000-0001-7935-9910, Lareau, Caleb A. ORCID: https://orcid.org/0000-0003-4179-4807, Liu, Ilon ORCID: https://orcid.org/0000-0002-6186-3630 and Ludwig, Leif S. ORCID: https://orcid.org/0000-0002-2916-2164
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| 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 |
| Related to: |
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