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Nuclei isolation of multiple brain cell types for omics interrogation

Abstract

We present a nuclei isolation protocol for genomic and epigenomic interrogation of multiple cell type populations in the human and rodent brain. The nuclei isolation protocol allows cell type-specific profiling of neurons, microglia, oligodendrocytes, and astrocytes, being compatible with fresh and frozen samples obtained from either resected or postmortem brain tissue. This 2-day procedure consists of tissue homogenization with fixation, nuclei extraction, and antibody staining followed by fluorescence-activated nuclei sorting (FANS) and does not require specialized skillsets. Cell type-specific nuclei populations can be used for downstream omic-scale sequencing applications with an emphasis on epigenomic interrogation such as histone modifications, transcription factor binding, chromatin accessibility, and chromosome architecture. The nuclei isolation protocol enables translational examination of archived healthy and diseased brain specimens through utilization of existing medical biorepositories.

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Fig. 1: Schematic of cell type-specific nuclei isolation from frozen brain tissue.
Fig. 2: FANS gating strategy for sorting of cell type-specific nuclei populations.
Fig. 3: Enhancer atlases from ex vivo microglia and PU.1 nuclei.

Data availability

The processed ATAC-seq and H3K27ac ChIP-seq tracks for the Schwannoma myeloid cells has been made available as part of a previously published UCSC genome browser session (hg19) containing ATAC-seq, ChIP-seq, and PLAC-seq datasets for brain cell types1: https://genome.ucsc.edu/s/nottalexi/glassLab_BrainCellTypes_hg19. ATAC-seq and ChIP-seq datasets for ex vivo microglia and brain-derived PU.1 nuclei were previously published1,28 and are available on dbGap (https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs001373.v2.p2).

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Acknowledgements

We thank M. P. Pasillas for technical assistance and scientific discussions. A.N. was supported by the Alzheimer’s Association (grant no. AARF-18-531498), the Altman Clinical & Translational Research Institute at UCSD (National Center for Advancing Translational Sciences, supported by NIH grant no. KL2TR001444-6), a pilot project grant from UCSD Shiley-Marcos ADRC 1P30AG062429, and the UK Dementia Research Institute, which receives its funding from UK DRI Ltd, funded by the UK Medical Research Council, Alzheimer’s Society, and Alzheimer’s Research UK. J.C.M.S. was supported by the Interdisciplinary Research Fellowship in NeuroAIDS (NIH R25MH081482-12) and the HNRC CSPAR Developmental Core award (NIH 5P30MH062512-18). B.R.F. was supported by the National Institutes of Health (NIH) grant, 1F30AG062159-01. These studies were carried out with grant support to C.K.G. from the NIH R01 NS096170, R01 AG056511, and R01 AG061060-01, and from the Cure Alzheimer’s Fund Gifford Neuroinflammation Consortium CAF 20183159.

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Authors and Affiliations

Authors

Contributions

A.N., J.C.M.S., and C.K.G. conceptualized the study; A.N. and J.C.M.S. optimized the methodology with input from B.R.F.; A.N. acquired and analyzed nuclei ChIP-seq and ATAC-seq datasets; A.N., J.C.M.S., B.R.F., and C.K.G. wrote the manuscript.

Corresponding authors

Correspondence to Alexi Nott, Johannes C. M. Schlachetzki or Christopher K. Glass.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Dimitrios Davalos, Elvira Mass, and Cindy van Velthoven for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Nott, A. et al. Science 366, 1134−1139 (2019): https://doi.org/10.1126/science.aay0793

Breuss, M. W. et al. Preprint at bioRxiv (2020): https://doi.org/10.1101/2020.08.10.244814

Key data used in this protocol

Nott, A. et al. Science 366, 1134-1139 (2019): https://doi.org/10.1126/science.aay0793

Gosselin, D. et al. Science 356, eaal3222 (2017): https://doi.org/10.1126/science.aal3222

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Nott, A., Schlachetzki, J.C.M., Fixsen, B.R. et al. Nuclei isolation of multiple brain cell types for omics interrogation. Nat Protoc 16, 1629–1646 (2021). https://doi.org/10.1038/s41596-020-00472-3

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