Extraction and Purification of Single Nuclei from Frozen Human Brain Tissue.
Cell diversity
Epigenomics
Extraction
FANS
Genomic mosaicism
Human brain nuclei
Single nucleus
Transcriptomics
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2023
2023
Historique:
entrez:
18
11
2022
pubmed:
19
11
2022
medline:
23
11
2022
Statut:
ppublish
Résumé
Resolving the complexity of the human brain at the level of single cells is essential to gaining an understanding of the immense diversity of cell types and functional states in both healthy and diseased brains. To exploit fully the technologies available for such studies, one must extract and isolate pure nuclei from unfixed postmortem tissue while preserving the molecules to be interrogated. Currently, nuclei are necessary substitutes for individual brain cells, since myriad cell types/sub-types constituting the human brain are embedded within the neuropil-a complex milieu of interconnected cells, processes, and synapses-which precludes intact and selective isolation of single brain cells. Here, we describe a protocol for the extraction and purification of intact single nuclei from frozen human brain tissue along with modifications to accommodate numerous downstream analyses, particularly for transcriptomic applications.
Identifiants
pubmed: 36399263
doi: 10.1007/978-1-0716-2655-9_2
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
31-42Subventions
Organisme : NIA NIH HHS
ID : R01 AG065541
Pays : United States
Organisme : NIA NIH HHS
ID : R01 AG071465
Pays : United States
Organisme : NIA NIH HHS
ID : R56 AG073965
Pays : United States
Informations de copyright
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
Références
Rehen SK et al (2005) Constitutional aneuploidy in the normal human brain. J Neurosci 25(9):2176–2180
doi: 10.1523/JNEUROSCI.4560-04.2005
pubmed: 15745943
pmcid: 6726097
Rehen SK et al (2001) Chromosomal variation in neurons of the developing and adult mammalian nervous system. Proc Natl Acad Sci U S A 98(23):13361–13366
doi: 10.1073/pnas.231487398
pubmed: 11698687
pmcid: 60876
Bushman DM et al (2015) Genomic mosaicism with increased amyloid precursor protein (APP) gene copy number in single neurons from sporadic Alzheimer’s disease brains. elife 4:e05116
doi: 10.7554/eLife.05116
pmcid: 4337608
Kaushal D et al (2003) Alteration of gene expression by chromosome loss in the postnatal mouse brain. J Neurosci 23(13):5599–5606
doi: 10.1523/JNEUROSCI.23-13-05599.2003
pubmed: 12843262
pmcid: 6741244
Westra JW et al (2010) Neuronal DNA content variation (DCV) with regional and individual differences in the human brain. J Comp Neurol 518(19):3981–4000
doi: 10.1002/cne.22436
pubmed: 20737596
pmcid: 2932632
Westra JW, Barral S, Chun J (2009) A reevaluation of tetraploidy in the Alzheimer’s disease brain. Neurodegener Dis 6(5–6):221–229
doi: 10.1159/000236901
pubmed: 19738367
pmcid: 2837890
Rohrback S et al (2018) Genomic mosaicism in the developing and adult brain. Dev Neurobiol 78(11):1026–1048
doi: 10.1002/dneu.22626
pubmed: 30027562
pmcid: 6214721
Rohrback S et al (2018) Submegabase copy number variations arise during cerebral cortical neurogenesis as revealed by single-cell whole-genome sequencing. Proc Natl Acad Sci U S A 115(42):10804–10809
doi: 10.1073/pnas.1812702115
pubmed: 30262650
pmcid: 6196524
Lee MH et al (2018) Somatic APP gene recombination in Alzheimer’s disease and normal neurons. Nature 563(7733):639–645
doi: 10.1038/s41586-018-0718-6
pubmed: 30464338
pmcid: 6391999
Lake BB et al (2018) Integrative single-cell analysis of transcriptional and epigenetic states in the human adult brain. Nat Biotechnol 36(1):70–80
doi: 10.1038/nbt.4038
pubmed: 29227469
Lake BB et al (2017) A comparative strategy for single-nucleus and single-cell transcriptomes confirms accuracy in predicted cell-type expression from nuclear RNA. Sci Rep 7(1):6031
doi: 10.1038/s41598-017-04426-w
pubmed: 28729663
pmcid: 5519641
Lake BB et al (2016) Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain. Science 352(6293):1586–1590
doi: 10.1126/science.aaf1204
pubmed: 27339989
pmcid: 5038589
Bakken TE et al (2018) Single-nucleus and single-cell transcriptomes compared in matched cortical cell types. PLoS One 13(12):e0209648
doi: 10.1371/journal.pone.0209648
pubmed: 30586455
pmcid: 6306246
Krishnaswami SR et al (2016) Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons. Nat Protoc 11(3):499–524
doi: 10.1038/nprot.2016.015
pubmed: 26890679
pmcid: 4941947
Zaghlool A et al (2021) Characterization of the nuclear and cytosolic transcriptomes in human brain tissue reveals new insights into the subcellular distribution of RNA transcripts. Sci Rep 11(1):4076
doi: 10.1038/s41598-021-83541-1
pubmed: 33603054
pmcid: 7893067
Palmer CR et al (2021) Altered cell and RNA isoform diversity in aging Down syndrome brains. Proc Natl Acad Sci U S A 118(47):e2114326118
doi: 10.1073/pnas.2114326118
pubmed: 34795060
pmcid: 8617492
Bakken TE et al (2021) Comparative cellular analysis of motor cortex in human, marmoset and mouse. Nature 598(7879):111–119
doi: 10.1038/s41586-021-03465-8
pubmed: 34616062
pmcid: 8494640
Hodge RD et al (2019) Conserved cell types with divergent features in human versus mouse cortex. Nature 573(7772):61–68
doi: 10.1038/s41586-019-1506-7
pubmed: 31435019
pmcid: 6919571