Abstract
Single-cell and single-nucleus sequencing techniques are a burgeoning field with various biological, biomedical and clinical applications. Numerous high- and low-throughput methods have been developed for sequencing the RNA and DNA content of single cells. However, for all these methods, the key requirement is high-quality input of a single-cell or single-nucleus suspension. Preparing such a suspension is the limiting step when working with fragile, archived tissues of variable quality. This hurdle can prevent such tissues from being extensively investigated with single-cell technologies. We describe a protocol for preparing single-nucleus suspensions within the span of a few hours that reliably works for multiple postmortem and archived tissue types using standard laboratory equipment. The stages of the protocol include tissue preparation and dissociation, nuclei extraction, and nuclei concentration assessment and capture. The protocol is comparable to other published protocols but does not require fluorescence-assisted nuclei sorting (FANS) or ultracentrifugation. The protocol can be carried out by a competent graduate student familiar with basic laboratory techniques and equipment. Moreover, these preparations are compatible with single-nucleus (sn)RNA-seq and assay for transposase-accessible chromatin (ATAC)-seq using the 10X Genomics Chromium system. The protocol reliably results in efficient capture of single nuclei for high-quality snRNA-seq libraries.
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Data availability
Raw sequencing data are accessible on GEO using the accession number GSE144136.
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Acknowledgements
G.T. holds a Canada Research Chair (Tier 1) and a NARSAD Distinguished Investigator Award. He is supported by grants from the Canadian Institute of Health Research (CIHR) (FDN148374 and EGM141899). Further support is provided by CFI Leaders Opportunity Fund, CFI#33408 and #35444, Genome Canada Science Genome Innovation Node grant (to J.R.). C. Nascimento was supported by grant 2018/11963-8 from the São Paulo Research Foundation (FAPESP). M.S. is hosted within the MRC Integrative Epidemiology Unit at the University of Bristol, which is supported by The Medical Research Council (MC_UU_00011/5). We acknowledge the expert help of the Douglas-Bell Canada Brain Bank staff (J. Prud’homme, M. Bouchouka and A. Baccichet), and the technology development team at the McGill University and Genome Quebec Innovation Centre. The Douglas-Bell Canada Brain Bank is supported in part by funding from the Canada First Research Excellence Fund, awarded to McGill University for the Healthy Brains for Healthy Lives project, and from the Fonds de recherche du Québec - Santé (FRQS) through the Quebec Network on Suicide, Mood Disorders and Related Disorders. The present study used the services of the Molecular and Cellular Microscopy Platform (MCMP) at the Douglas Institute.
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M.M. and C. Nagy developed nucleus extraction protocol, prepared nuclei and wrote the manuscript. Y.C.W. preformed 10X snRNA-seq protocol. C. Nascimento performed 10X snATAC-seq protocol. A.C. performed snATAC-seq data analysis. M.S. and J.F.T. guided bioinformatic analysis. N.M. contributed to tissue processing and data interpretation. J.R. provided technical single-cell expertise and experimental support. G.T. provided general oversight, including in experimental design. All authors contributed to manuscript preparation.
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Peer review information Nature Protocols thanks Rebecca D. Hodge and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol
Jessa, S. et al. Nat. Genet. 51, 1702–1713 (2019): https://doi.org/10.1038/s41588-019-0531-7
Reiner, B. C. et al. Preprint at bioRxiv (2020): https://doi.org/10.1101/2020.07.29.227355
Nagy, C. et al. Nat. Neurosci. 23, 771–781 (2020): https://doi.org/10.1038/s41593-020-0621-y
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Maitra, M., Nagy, C., Chawla, A. et al. Extraction of nuclei from archived postmortem tissues for single-nucleus sequencing applications. Nat Protoc 16, 2788–2801 (2021). https://doi.org/10.1038/s41596-021-00514-4
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DOI: https://doi.org/10.1038/s41596-021-00514-4
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