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Cited by 9Single-cell and spatial transcriptomicsGenomics and Phylogenetic StudiesMolecular Biology Techniques and Applications

Comparative analysis of nuclei isolation methods for brain single-nucleus RNA sequencing

Open access
Kersey, Holly N., Acri, Dominic J., Dabin, Luke C., Hartigan, Kelly A., Mustaklem, Richard, Park, Jung Hyun, Kim, Jungsu·2026·Cell Reports Methods
DOI 10.1016/j.crmeth.2026.101337PMC PMC11974938

Published version of a preprint

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Abstract

Single-nucleus RNA sequencing (snRNA-seq) enables resolving cellular heterogeneity in complex tissues. snRNA-seq overcomes limitations of traditional single-cell RNA-seq by using nuclei instead of cells, allowing to utilize frozen tissues and difficult-to-isolate cell types. Although various nuclei isolation methods have been developed, systematic evaluations of their effects on nuclear integrity and subsequent data quality remain lacking, a critical gap with profound implications for the rigor and reproducibility. To address this, we compared three mechanistically distinct nuclei isolation strategies with brain tissues: a sucrose gradient centrifugation-based method, a spin column-based method, and a machine-assisted platform. All methods successfully captured diverse cell types but revealed considerable protocol-dependent differences in cell type proportions, transcriptional homogeneity, and the preservation of cell-type-specific and cell-state-specific markers. Moreover, isolation workflows differentially influenced contamination levels from ambient, mitochondrial, and ribosomal RNAs. Our findings establish nuclei isolation methodology as a critical experimental variable shaping snRNA-seq data quality and biological interpretation. MOTIVATION: Single-nucleus RNA sequencing (snRNA-seq) has become an essential tool for transcriptomic analysis of complex tissues. However, the quality and efficiency of data generation depend heavily on the method used for nuclear isolation. The existing isolation techniques vary in their ability to preserve nuclear integrity, minimize ambient RNA contamination, and optimize recovery rates. Despite these differences in quality, a systematic comparison of these methods, specifically for brain tissue, is lacking. This gap poses a challenge for researchers in choosing the most suitable approach for their particular experimental requirements. To address this critical issue, our study directly compared three nuclei isolation methods and evaluated their performance in terms of yield, purity, and downstream sequencing quality. By providing a comprehensive assessment, we aim to guide researchers in selecting the most appropriate isolation protocol for their snRNA-seq experiments, ensuring optimal results and advancing the study of complex brain tissues at the single-nucleus level.

AI snapshot

Written from the paper’s full text.

TL;DR

This study compared three mechanistically distinct nuclei isolation methods for brain snRNA-seq: sucrose gradient centrifugation, spin column-based isolation, and a machine-assisted platform. It found protocol-dependent differences in nuclei yield, integrity, contamination, cell-type proportions, and transcriptional/marker signatures, with the machine-assisted method generally showing the highest consistency and lowest mitochondrial/ribosomal contamination.

Key findings

  • Centrifugation-based and machine-assisted methods yielded similar nuclei numbers: ~2 million nuclei or ~60,000 nuclei/mg input, whereas the column-based method yielded 25% fewer. Intact nuclei were ~100% for machine-assisted, 85% for centrifugation-based, and 35% for column-based.
  • Across 98,452 captured nuclei and 36 clusters, most nuclei were excitatory neurons (53.9%) and inhibitory neurons (17.2%). Centrifugation-based isolation captured the largest astrocyte proportion (13.9%), while machine-assisted isolation captured the largest microglia (5.6%) and oligodendrocyte (15.9%) proportions.
  • Quality-control metrics differed by method: column-based samples had inconsistent doublet rates (3.2%–5.7%) and elevated SoupX ambient RNA rho values (>0.25), nearly twofold higher than centrifugation- and machine-assisted methods; machine-assisted rho was ~0.14. Machine-assisted samples had <0.5% mitochondrial-derived reads, versus 1–3% for centrifugation-based and 2–4% for column-based methods.
  • Glial marker and state signatures varied: machine-assisted isolation showed the highest microglia-specific marker enrichment and negligible Plp1 expression in microglia, while centrifugation-based isolation showed Mbp expression and highest Glul/Ptgds expression. Machine-assisted microglia had the highest homeostatic and lowest disease-associated signature scores.

Methods

  • Cortical tissue from 6-month-old male C57BL/6J mice was used, with N=2 per isolation method and ~30 mg anterior cortex input per method. The three methods were manual homogenization followed by sucrose gradient centrifugation, a 10x Genomics Chromium Nuclei Isolation spin column method, and the Singulator 100 machine-assisted platform.
  • snRNA-seq libraries were prepared with 10x Genomics Chromium Next GEM Single Cell 3′ v3.1 and sequenced on NovaSeq 6000 to a mean depth of 437.9 ± 23.9 million reads per sample.
  • Data were processed with Cell Ranger v7.0.1 aligned to GRCm38, then analyzed with SoupX, DoubletFinder, Seurat/SCTransform, scMCA/PanglaoDB, speckle, ROGUE, and UCell.

Limitations

  • The paper states that six samples were sequenced across three methods, that nuclei preparations were repeated multiple times to ensure representative sequenced samples, and that the study focused on mouse cortical tissue under normal physiological conditions, so findings may not fully represent other brain regions, tissues, species, or pathological states.
  • Evident from the design: N=2 per method limits statistical power and reproducibility assessment; only male mice at 6 months and one brain region were tested; and method-specific differences may partly reflect protocol implementation or optimization rather than inherent method class.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.crmeth.2026.101337

Cites

64 of 64 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

26 of 30 cited sources checked · 6 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    Use of nuclei enables study of postmortem, frozen, or biobank tissue, including neurodegenerative conditions such as Alzheimer's disease.

    “Additionally, the use of nuclei, rather than whole cells, enables the study of samples that were previously difficult to analyze via scRNA-seq, such as postmortem, frozen, or biobank tissue, including those obtained from individuals with neurodegenerative conditions such as Alzheimer’s disease (AD)[12, 16].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [12] Kim, 2023, Perspectives on single-nucleus RNA sequencing in different cell types and tissues, J. Pathol. Transl. Med. source

      The Experimental Procedures section says archived frozen tissue is a primary target for single-nucleus RNA sequencing, and “Neurons in the brain” describes its use with postmortem human brain tissue. That section also discusses neurodegenerative disease, but the copy does not explicitly establish the claimed use of biobank tissue from individuals with Alzheimer’s disease; Alzheimer’s appears only in a cited study’s title in the References. Page numbers were not available in our copy. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [16] Maitra, 2021, Extraction of nuclei from archived postmortem tissues for single-nucleus sequencing applications, Nat. Protoc. source
  • Premisecitation

    Low-quality nuclei isolation protocols can introduce artifacts that compromise data interpretation and experimental reliability.

    “Low-quality nuclei isolation protocols can introduce artifacts that compromise the proper data interpretation and the experimental reliability[32, 33].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [32] Hong, 2022, Comprehensive generation, visualization, and reporting of quality control metrics for single-cell RNA sequencing data, Nat. Commun. source

      The Introduction says that sample-preparation artifacts in single-cell RNA sequencing can confound downstream analyses or produce erroneous findings. It discusses cell dissociation and other cell-based procedures, not low-quality nuclei isolation protocols specifically, so the claim extends beyond the source's stated setting. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [33] Machado, 2021, Stress relief: emerging methods to mitigate dissociation-induced artefacts, Trends Cell Biol. source
  • Premisecitation

    Ambient RNA from lysed cells can lead to false positives in gene expression profiles, obscuring true cell type-specific markers.

    “For example, ambient RNA from lysed cells, a ubiquitous issue of scRNA- and snRNA-seq datasets, can lead to false positives in gene expression profiles, obscuring true cell type-specific markers[34, 35].”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [34] Caglayan, 2022, Neuronal ambient RNA contamination causes misinterpreted and masked cell types in brain single-nuclei datasets, Neuron source

      The Introduction (page unavailable) says freely floating transcripts contaminate endogenous expression profiles and can skew them enough to cause biological misinterpretation. The Results section on glial nuclei (page unavailable) reports ambient RNA markers appearing falsely expressed in those nuclei, supporting the concern about misleading cell-type profiles. Our copy does not establish that the RNA specifically comes from lysed cells, and the paper’s findings focus on brain single-nuclei datasets rather than demonstrating the claim across scRNA- and snRNA-seq datasets generally. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [35] Zhang, 2023, Ambient RNAs removal of cortex-specific snRNA-seq reveals Apoe(+) microglia/macrophage after deeper cerebral hypoperfusion in mice, J. Neuroinflammation source
  • Interpretationcitation

    Ambient RNA contamination may distort transcriptomic profiles, leading to the underrepresentation of specific cell types or misclassification of cellular states.

    “This contamination may distort transcriptomic profiles, leading to the underrepresentation of specific cell types or misclassification of cellular states[34, 37].”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [34] Caglayan, 2022, Neuronal ambient RNA contamination causes misinterpreted and masked cell types in brain single-nuclei datasets, Neuron source
    • cites [37] Young, 2020, SoupX removes ambient RNA contamination from droplet-based single-cell RNA sequencing data, GigaScience source

      The Introduction (page not available) says ambient RNA can confound biological interpretation. In “Ambient RNA Confounds Interpretation in Complex Experiments” (page not available), correction changed many cells’ cluster assignments, and the authors describe how contaminating expression could lead to false conclusions about cells. These passages support distorted profiles and possible misclassification, but do not establish underrepresentation of specific cell types. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Premisecitation

    Many cell types, such as microglia, are especially sensitive to dissociation-induced activation.

    “Many cell types, such as microglia, are especially sensitive to dissociation-induced activation, which is a known challenge in single-cell studies[9, 10].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [9] van den Brink, 2017, Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations, Nat. Methods source
    • cites [10] Marsh, 2022, Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain, Nat. Neurosci. source

      The Results section ('Mouse microglia are especially sensitive to ex vivo alterations'; page not provided) shows that enzymatic dissociation induced an artifactual activation signature in microglia. But it reports that microglia/myeloid cells were the only major CNS cell class with a clear inhibitor-dependent clustering response; other cell types showed only slight changes. Thus the source supports the microglia example and the single-cell-study challenge, not the claim that many cell types are especially sensitive. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • May combine two claims. 22
  • Classified as probably not a substantial claim. 7

  • May combine two claims.

    Premisecitation

    snRNA-seq overcomes the need for intact cell dissociation, thereby minimizing dissociation-associated challenges and providing a more physiologically accurate representation of the brain's cellular heterogeneity.

    “snRNA-seq overcomes the need for intact cell dissociation, thereby minimizing dissociation-associated challenges and providing a more physiologically accurate representation of the brain’s cellular heterogeneity[12, 14, 15].”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [12] Kim, 2023, Perspectives on single-nucleus RNA sequencing in different cell types and tissues, J. Pathol. Transl. Med. source

      The Abstract says snRNA-seq avoids enzymatic dissociation of intact cells and can reduce cellular and stress-response biases. The “Neurons in the brain” section says it improves representation of neuronal cell types, but also notes limitations in capturing non-neuronal cells and recommends combining it with scRNA-seq. The source therefore does not establish that snRNA-seq alone gives a more physiologically accurate representation of the brain’s overall cellular heterogeneity. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [14] Lake, 2016, Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain, Science source

      The Main Text (page unavailable) says isolating nuclei reduces contamination or degradation encountered with whole-neuron dissociation, and reports that the method identifies neuronal subtypes. It does not establish that snRNA-seq gives a more physiologically accurate representation of the brain’s overall cellular heterogeneity; this study examined neuronal nuclei from six cortical regions. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [15] Tran, 2021, Single-nucleus transcriptome analysis reveals cell-type-specific molecular signatures across reward circuitry in the human brain, Neuron source

      The Methods describe sequencing nuclei isolated from frozen brain tissue, supporting the point that intact cell dissociation was not required. The paper does not establish that this minimizes dissociation-associated challenges or provides a more physiologically accurate representation of cellular heterogeneity; the Discussion instead notes incomplete transcriptomic capture and that some cell populations were not captured by the protocols. Page numbers were not available in our copy. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    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].”
    Quote in the paperSubstantial claim5 of 6 sources checked
    • cites [6] Waag, 2023, Single-Nucleus RNA-Sequencing in Brain Tissue, Curr. Protoc. source

      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)

      Experimental — accuracy not yet measured
    • cites [8] Lacar, 2016, Nuclear RNA-seq of single neurons reveals molecular signatures of activation, Nat. Commun. source

      The paper’s Abstract (page not provided) says dendritic loss is likely during protease dissociation, supporting the concern that conventional preparation can compromise neuronal integrity. It does not identify mechanical shearing as the cause or say that dissociation activates glia; its Discussion instead reports dissociation-induced gene expression in neurons. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [9] van den Brink, 2017, Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations, Nat. Methods source
    • cites [10] Marsh, 2022, Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain, Nat. Neurosci. source

      The Discussion reports that typical enzymatic digestion of mouse brain tissue induces ex vivo transcriptional changes, preferentially in microglia and other brain myeloid cells, supporting the glial-activation part. It also reports depletion of other CNS cell types with a mechanical Dounce-and-Percoll protocol, but does not establish that mechanical shearing compromises neuronal integrity. Page numbers were not provided in our copy. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [11] Lake, 2017, A comparative strategy for single-nucleus and single-cell transcriptomes confirms accuracy in predicted cell-type expression from nuclear RNA, Sci. Rep. source

      The Discussion says the stress of whole-cell dissociation may influence gene expression, and the Introduction describes the difficulty of obtaining intact, viable cells from brain tissue. Neither passage identifies mechanical shearing as a cause of neuronal damage or says dissociation induces glial activation; searches for those specific points found no supporting passage in our copy. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [12] Kim, 2023, Perspectives on single-nucleus RNA sequencing in different cell types and tissues, J. Pathol. Transl. Med. source

      The Abstract says enzymatic tissue dissociation may damage neurons, and the Experimental Procedures section describes neurons as fragile and difficult to dissociate. The paper does not attribute neuronal damage to mechanical shearing or say that dissociation induces glial activation. No page numbers were supplied for these passages. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Excessive mitochondrial and ribosomal RNA can overwhelm sequencing data, reducing depth of informative transcripts and hindering detection of low-abundance genes.

    “Excessive levels of contaminating mitochondrial and ribosomal RNA can overwhelm sequencing data, reducing the depth of informative transcripts and hindering the detection of low-abundance genes[36].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [36] Alvarez, 2020, Enhancing droplet-based single-nucleus RNA-seq resolution using the semi-supervised machine learning classifier DIEM, Sci. Rep. source

      The Introduction and Results (pages not provided) identify mitochondrial RNA as background contamination and report that it can bias expression and drive spurious clusters. They do not establish that ribosomal RNA contamination overwhelms sequencing data, reduces informative-transcript depth, or hinders detection of low-abundance genes; a search for “ribosomal” found no supporting passage. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    All three methods typically involve enzymatic digestion, which can potentially activate certain cell types, introducing a common source of potential bias.

    “Notably, all three methods typically involve enzymatic digestion, which can potentially activate certain cell types, introducing a common source of potential bias[10].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [10] Marsh, 2022, Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain, Nat. Neurosci. source

      The Discussion reports that typical enzymatic digestion of mouse brain tissue induced transcriptional changes, preferentially in microglia and other brain myeloid cells, and could confound downstream analysis. It says enzymatic dissociation is used in the majority of single-cell studies of CNS populations, but does not establish that each of the citing sentence’s three unspecified methods typically involves it. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A decrease in astrocytic Glul expression has been associated with epilepsy and subsequent neurodegeneration.

    “Also, a decrease in astrocytic Glul expression has been associated with epilepsy and subsequent neurodegeneration[52, 53].”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [52] Eid, 2019, Astrocytes and Glutamine Synthetase in Epileptogenesis, J. Neurosci. Res. source
    • cites [53] Sandhu, 2021, Astroglial Glutamine Synthetase and the Pathogenesis of Mesial Temporal Lobe Epilepsy, Front. Neurol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Tmem119 expression is significantly reduced in activated microglia in AD.

    “For instance, Tmem119 expression is significantly reduced in activated microglia in AD, whereas C1qa is associated with microglial activation after traumatic brain injury[49–51].”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [49] Holden, 2021, Complement factor C1q mediates sleep spindle loss and epileptic spikes after mild brain injury, Science source

      The cited paper’s “Microglia are a source of C1q in the thalamus” section examines C1qa after mild traumatic brain injury, not Tmem119 expression in Alzheimer’s disease. A search for Tmem119 and activated microglia in AD found no passage addressing the claimed reduction. The paper supports the citing sentence’s separate C1qa/TBI clause, but not the quoted Tmem119/AD claim. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [50] Yan, 2022, Elevated Serum Complement C1q Levels After Traumatic Brain Injury and Its Association with Poor Prognosis, Neuropsychiatr. Dis. Treat. source
    • cites [51] Ma, 2024, Tmem119 expression is downregulated in a subset of brain metastasis-associated microglia, BMC Neurosci. source

      The Results section reports significantly reduced Tmem119 expression in microglia activated by LPS, and the Discussion reports a reduction in brain metastasis-associated microglia. The Background says TMEM119 was detected in post-mortem Alzheimer’s disease samples, but the paper does not establish a significant reduction in activated microglia in AD. Page numbers are unavailable in our copy. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    C1qa is associated with microglial activation after traumatic brain injury.

    “For instance, Tmem119 expression is significantly reduced in activated microglia in AD, whereas C1qa is associated with microglial activation after traumatic brain injury[49–51].”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [49] Holden, 2021, Complement factor C1q mediates sleep spindle loss and epileptic spikes after mild brain injury, Science source
    • cites [50] Yan, 2022, Elevated Serum Complement C1q Levels After Traumatic Brain Injury and Its Association with Poor Prognosis, Neuropsychiatr. Dis. Treat. source

      The Discussion reports increased C1q expression after experimental traumatic brain injury, localized in part to microglia, and describes C1q effects on cultured microglial cells. It does not establish that C1qa expression is associated specifically with microglial activation after traumatic brain injury; the paper's own TBI measurements concern serum C1q. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [51] Ma, 2024, Tmem119 expression is downregulated in a subset of brain metastasis-associated microglia, BMC Neurosci. source

      The Background discusses TMEM119 detection in traumatic brain injury, and the Discussion links loss of Tmem119 to microglial activation. A search for C1qa found no passage connecting it to microglial activation after traumatic brain injury in our copy. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    The centrifugation-based method yielded the greatest number of nuclei but also exhibited pronounced transcriptional heterogeneity.

    “The centrifugation-based method yielded the greatest number of nuclei but also exhibited pronounced transcriptional heterogeneity, likely due to differences in RNA degradation or cytoplasmic contamination between individual preparations (Figures 1, 2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Siglech showed robust expression across all methods but greater expression in the centrifugation-based and machine-assisted methods.

    “Two other microglia-specific markers, Hexb and Siglech showed robust expression across all methods but was greater expression in the centrifugation-based and machine-assisted methods (Figure 4C, D).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The centrifugation-based method produced defined individual nuclei with minimal background debris.

    “The centrifugation-based method produced defined individual nuclei with minimal background debris (Figure 1B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In total, 98,452 nuclei were captured from the six samples, forming 36 distinct clusters.

    “In total, 98,452 nuclei were captured from the six samples, forming 36 distinct clusters (Figure 2A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The machine-assisted method yielded well-separated, intact nuclei with negligible debris.

    “The machine-assisted method yielded well-separated, intact nuclei with negligible debris (Figure 1D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Hexb showed robust expression across all methods but greater expression in the centrifugation-based and machine-assisted methods.

    “Two other microglia-specific markers, Hexb and Siglech showed robust expression across all methods but was greater expression in the centrifugation-based and machine-assisted methods (Figure 4C, D).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Sequencing was performed to a mean sequencing depth of 437.9 +/− 23.9 million reads per sample.

    “Sequencing was performed on a NovaSeq 6000 (v1.5 S2; Illumina) with a 28–10-10–91 read setup to a mean sequencing depth of 437.9 +/− 23.9 million reads per sample.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingreasoning

    Each nuclear isolation method significantly affects data quality, cell type representation, and transcriptional integrity.

    “Each method significantly affects data quality, cell type representation, and transcriptional integrity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Machine-assisted isolation maximizes microglia-specific signals while minimizing oligodendrocyte marker contamination.

    “For example, machine-assisted isolation maximizes microglia-specific signals while minimizing oligodendrocyte marker contamination.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Compared with the column-based approach, the centrifugation-based and machine-assisted methods are more effective in terms of both nuclear yield and purity.

    “These findings demonstrate that, compared with the column-based approach, the centrifugation-based and machine-assisted methods are more effective in terms of both nuclear yield and purity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The machine-assisted method yielded nuclei preparations with high consistency between samples and low contamination levels, whereas the column-based and centrifugation-based methods presented greater sample-to-sample variability and elevated contamination levels.

    “The results indicate that the machine-assisted method yielded nuclei preparations with high consistency between samples and low contamination levels, whereas the column-based and centrifugation-based methods presented greater sample-to-sample variability and elevated contamination levels.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: High-quality nuclei preparations should contain minimal to no cytoplasmic content.

  • May combine two claims.

    Interpretationown data

    All three nuclear isolation methods successfully capture diverse cell types from mouse cortical tissue, but exhibit differences in cell type proportions and transcriptional homogeneity.

    “These data demonstrate that although all three nuclear isolation methods successfully capture diverse cell types from mouse cortical tissue, they exhibit differences in cell type proportions and transcriptional homogeneity.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The uniformity of identified cell populations is a crucial quality control metric for accurate cell type identification.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All animal experiments were approved and performed in compliance with the guidelines of the Institutional Animal Care and Use Committee at Indiana University.

    “All animal experiments were approved and performed in compliance with the guidelines of the Institutional Animal Care and Use Committee at Indiana University.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The machine-assisted method preserved the strongest expression of homeostatic microglial markers and the most accurate representation of their transcriptional signatures.

    “When microglial populations were examined, the machine-assisted method preserved the strongest expression of homeostatic microglial markers and the most accurate representation of their transcriptional signatures (Figure 4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The machine-assisted method resulted in the highest nuclei viability and the lowest levels of mitochondrial and ribosomal RNA contamination.

    “In contrast, the machine-assisted method resulted in the highest nuclei viability and the lowest levels of mitochondrial and ribosomal RNA contamination (Figures 1, 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Such artifacts can lead to erroneous classification of microglial states, potentially confounding understanding of neuroinflammatory processes in AD and other neurodegenerative diseases.

    “Such artifacts can lead to erroneous classification of microglial states, potentially confounding our understanding of neuroinflammatory processes in AD and other neurodegenerative diseases.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

    Study design and conductnone given

    The proposed approach provides confidence in the results despite ongoing debate about powering in single-cell experiments.

    “Although the powering of a single-cell experiment remains a topic of active debate in the field, the proposed approach provides confidence in the results.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Nuclei preparations were repeated multiple times, in some cases dozens of times, to ensure that the sequenced samples were representative of the expected isolation.

    “Nuclei preparations were repeated multiple times, in some cases dozens of times, to ensure that the sequenced samples were representative of the expected isolation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The machine-assisted method demonstrated greater consistency across samples in key quality control metrics, including doublet formation and ambient RNA contamination, whereas the centrifugation- and column-based methods exhibited high variability.

    “Additionally, the machine-assisted method demonstrated greater consistency across samples in key quality control metrics, including doublet formation and ambient RNA contamination, whereas the centrifugation- and column-based methods exhibited high variability.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: In snRNA-seq analysis, a consistent doublet rate is generally preferable because it indicates more reliable and reproducible sample processing.

  • May combine two claims.

    Interpretationreasoning

    The variability observed in centrifugation- and column-based methods compromises data quality, cell type representation, and transcriptional integrity.

    “This variability not only compromises data quality, cell type representation, and transcriptional integrity but also underscores the need for methodological transparency and rigorous validation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The column-based method yielded the lowest number of nuclei but also resulted in the highest levels of ambient RNA contamination.

    “The column-based method, while yielding the lowest number of nuclei, also resulted in the highest levels of ambient RNA contamination.”
    Quote in the paperSubstantial claim
28 of 28

  • The paper relies on this without support.

    A systematic comparison of nuclei isolation methods specifically for brain tissue is lacking.

    “Despite these differences in quality, a systematic comparison of these methods, specifically for brain tissue, is lacking.”

  • The paper relies on this without support.

    The uniformity of identified cell populations is a crucial quality control metric for accurate cell type identification.

    “The uniformity of identified cell populations is a crucial quality control matix for accurate cell type identification.”

  • The paper relies on this without support.

    High-quality nuclei preparations should contain minimal to no cytoplasmic content.

    “High-quality nuclei preparations should contain minimal to no cytoplasmic content.”

  • The paper relies on this without support.

    In snRNA-seq analysis, a consistent doublet rate is generally preferable because it indicates more reliable and reproducible sample processing.

    “In the context of snRNA-seq analysis, a consistent doublet rate is generally preferable as it indicates more reliable and reproducible sample processing.”

  • The paper relies on this without support.

    The effectiveness of a nuclear isolation method for snRNA-seq depends on several factors, including tissue type, processing conditions, and specific research questions.

    “The effectiveness of a nuclear isolation method for snRNA-seq depends on several factors, including tissue type, processing conditions, and specific research questions.”

  • The paper relies on this without support.

    Different pathological states can affect isolation efficacy and quality.

    “Different pathological states can affect isolation efficacy and quality.”
6 of 6

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Figure 4J is assigned to two different analyses

    Minor

    The text identifies Figure 4J as showing Ptgds expression in microglial nuclei and later as showing homeostatic astrocyte signature scores.

  • Column-based ribosomal contamination is reported both below 0.03% and at 0.036%

    Minor

    The column-based protocol is said to have ribosomal-derived reads below 0.03%, but its reported average is 0.036%, which exceeds that threshold.

2 of 2

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. Acri, 2024, neurogenetics/SAHA: ALL VERSIONS., Zenodo
  2. Korotkevich, 2021, Fast gene set enrichment analysis, bioRxiv

Cited by

Sorted by citations

Authors
Kersey, H. N., Acri, D. J., Dabin, L. C., Hartigan, K. A., Mustaklem, R., Park, J. H., & Kim, J.
Affiliations
  • Indiana University Indianapolis (US)
  • Indiana University Bloomington (US)
Corresponding author
Kim, Jungsu
ORCID
Venue
Cell Reports Methods
Publisher
Elsevier BV
Published
2026-03
Volume
6
Issue
3
Pages
101337
ISSN
2667-2375 (ISSN-L)
DOI
10.1016/j.crmeth.2026.101337
PMC
PMC11974938
OpenAlex
W7140084071
Cited by
8
References
63
Citations by year
2026: 6 · 2025: 2
FWCI
11.05
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Reports Methods (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed (publishedVersion)
  • IUScholarWorks (Indiana University) (submittedVersion, cc-by)
  • PubMed Central (submittedVersion, cc-by)
  • Europe PMC (PubMed Central) (submittedVersion, cc-by)
Funders
  • National Institute on Aging (R21 AG072738, T32AG071444, R01 AG071281, U01AG076804, R01 AG077829, RF1 AG074543)
  • Eli Lilly and Company
  • National Institutes of Health
  • Lilly Endowment
  • International Business Machines Corporation
  • Indiana University School of Medicine
  • Indiana University
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, nuclei isolation, brain tissue, sucrose density gradient centrifugation, nuclear integrity, data quality, cell type composition, reproducibility

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-17.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-17.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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