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Cited by 15Single-cell and spatial transcriptomicsGenomics and Phylogenetic StudiesMolecular Biology Techniques and Applications

Single‐Nucleus RNA‐Sequencing in Brain Tissue

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Waag, Rebecca, Bohacek, Johannes·2023·Current Protocols
DOI 10.1002/cpz1.919
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Abstract

Neuroscience research greatly benefits from single‐cell sequencing technologies, which can reveal transcriptional alterations on a cellular level. However, preparing single‐cell suspensions is technically challenging, requires experience, and has several limitations that can influence the transcriptional readout. Performing sequencing of single nuclei instead of single cells alleviates several of the challenges of sample preparation and highlights acute nuclear transcription. Here, we provide a protocol to prepare a nuclei suspension for single‐nucleus RNA‐sequencing for cell type‐specific transcriptional profiling of brain tissue using the 10x Genomics single‐cell gene expression assay. Furthermore, we highlight important aspects to consider during experimental design and data analysis. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of single‐nucleus suspension Basic Protocol 2: Preparation and sequencing of single‐nucleus libraries for RNA‐seq

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  • Conventional cell dissociation protocols often compromise neuronal integrity through mechanical shearing or induce glial activation.

    “For example, conventional cell dissociation protocols often compromise the integrity of neurons through mechanical shearing or induce the activation of glia[6, 8–12].”

    From Comparative analysis of nuclei isolation methods for brain single-nucleus RNA sequencing (2026) · cites this paper as [6]

    The Introduction (page unavailable) says obtaining viable cells from brain tissue is difficult, and Understanding Results (page unavailable) describes dissociation-induced activation of immediate-early genes in microglia. These passages support a dissociation-related glial activation artifact, but do not establish that conventional cell dissociation compromises neuronal integrity through mechanical shearing. (LLM, conf=0.86)

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Authors
Waag, R., & Bohacek, J.
Affiliations
University of Zurich (CH)
Corresponding author
Bohacek, Johannes
ORCID
Venue
Current Protocols
Publisher
Wiley
Published
2023-11
Volume
3
Issue
11
Pages
e919-e919
ISSN
2691-1299 (ISSN-L)
DOI
10.1002/cpz1.919
OpenAlex
W4388870446
Cited by
15
References
47
Citations by year
2026: 5 · 2025: 7 · 2024: 2 · 2023: 1
FWCI
1.34
Type
journal article
Language
en
Open access
Licence
Locations
  • Current Protocols (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • Repository for Publications and Research Data (ETH Zurich) (submittedVersion, cc-by-nc)
  • ETH Zürich Research Collection
Funders
  • Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (172889, 204372)
  • Eidgenössische Technische Hochschule Zürich
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
Keywords
single-nucleus RNA sequencing, brain tissue, nuclear transcription, experimental design, data analysis
MeSH terms
RNA-Seq, RNA, Sequence Analysis, RNA, Solitary Nucleus, Gene Expression Profiling

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