Abstract
Mitochondria are unique organelles of eukaryotic cells that carry their own multicopy number and circular genome. In most mammals, including humans and mice, the size of the chromosome is ~16,000 base pairs and unlike nuclear DNA, mitochondrial DNA (mtDNA) is not densely compacted. This results in mtDNA to be highly accessible for enzymes such as the Tn5 transposase, commonly used for accessible chromatin profiling of nuclear chromatinized DNA. Here, we describe a method for the concomitant sequencing of mtDNA and accessible chromatin in thousands of individual cells via the mitochondrial single-cell assay for transposase accessible chromatin by sequencing (mtscATAC-seq). Our approach extends the utility of existing scATAC-seq products and protocols as we (Nam et al, Nat Rev Genet 22:3–18, 2021) fix cells using formaldehyde to retain mitochondria and its mtDNA within its originating cell, (Buenrostro et al, Nat Methods 10:1213–1218, 2013) modify lysis conditions to permeabilize cells and mitochondria, and (Corces et al, Nat Methods 14:959–962, 2017) optimize bioinformatic processing protocols to collectively increase mitochondrial genome coverage for downstream analysis. Here, we discuss the essentials for the experimental and computational methodologies to generate and analyze thousands of multiomic profiles of single cells over the course of a few days, enabling the profiling of accessible chromatin and mtDNA genotypes to reconstruct clonal relationships and studies of mitochondrial genetics and disease.
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References
Nam AS, Chaligne R, Landau DA (2021) Integrating genetic and non-genetic determinants of cancer evolution by single-cell multi-omics. Nat Rev Genet 22:3–18
Buenrostro JD, Giresi PG, Zaba LC et al (2013) Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods 10:1213–1218
Corces MR, Trevino AE, Hamilton EG et al (2017) An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat Methods 14:959–962
Lareau CA, Ludwig LS, Muus C et al (2021) Massively parallel single-cell mitochondrial DNA genotyping and chromatin profiling. Nat Biotechnol 39:451–461
Walker MA, Lareau CA, Ludwig LS et al (2020) Purifying selection against pathogenic mitochondrial DNA in human T cells. N Engl J Med 383:1556–1563
Ludwig LS, Lareau CA, Ulirsch JC et al (2019) Lineage tracing in humans enabled by mitochondrial mutations and single-cell genomics. Cell 176:1325–1339.e22
Penter L, Gohil SH, Lareau C et al (2021) Longitudinal single-cell dynamics of chromatin accessibility and mitochondrial mutations in chronic lymphocytic leukemia mirror disease history. Cancer Discov 11:3048. https://doi.org/10.1158/2159-8290.CD-21-0276
Lareau CA, Ludwig LS, Sankaran VG (2019) Longitudinal assessment of clonal mosaicism in human hematopoiesis via mitochondrial mutation tracking. Blood Adv 3:4161–4165
Stuart T, Srivastava A, Madad S et al (2021) Multimodal single-cell chromatin analysis with Signac. Nat Methods (in press)
Fang R, Preissl S, Li Y et al (2021) Comprehensive analysis of single cell ATAC-seq data with SnapATAC. Nat Commun 12:1–15
Granja JM, Corces MR, Pierce SE et al (2021) ArchR is a scalable software package for integrative single-cell chromatin accessibility analysis. Nat Genet 53:403–411
Mimitou EP, Lareau CA, Chen KY et al (2021) Scalable, multimodal profiling of chromatin accessibility, gene expression and protein levels in single cells. Nat Biotechnol 39:1246. https://doi.org/10.1038/s41587-021-00927-2
Acknowledgments
L.S.L. is supported by an Emmy Noether fellowship by the German Research Foundation (DFG, LU 2336/2-1) and a Hector Research Career Development Award. C.A.L. is supported by a Stanford Science Fellowship and a Parker Institute of Cancer Immunotherapy Scholarship. We are grateful to the Ludwig lab for useful feedback in the generation of this protocol.
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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
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Ludwig, L.S., Lareau, C.A. (2023). Concomitant Sequencing of Accessible Chromatin and Mitochondrial Genomes in Single Cells Using mtscATAC-Seq. In: Marinov, G.K., Greenleaf, W.J. (eds) Chromatin Accessibility. Methods in Molecular Biology, vol 2611. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2899-7_14
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DOI: https://doi.org/10.1007/978-1-0716-2899-7_14
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