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Cited by 483Genomics and Chromatin DynamicsSingle-cell and spatial transcriptomicsEpigenetics and DNA Methylation

Single-cell CUT&Tag profiles histone modifications and transcription factors in complex tissues

Open access
Bartosovic, Marek, Kabbe, Mukund, Castelo-Branco, Gonçalo·2021·Nature Biotechnology
DOI 10.1038/s41587-021-00869-9PMC PMC7611252
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Abstract

In contrast to single-cell approaches for measuring gene expression and DNA accessibility, single-cell methods for analyzing histone modifications are limited by low sensitivity and throughput. Here, we combine the CUT&Tag technology, developed to measure bulk histone modifications, with droplet-based single-cell library preparation to produce high-quality single-cell data on chromatin modifications. We apply single-cell CUT&Tag (scCUT&Tag) to tens of thousands of cells of the mouse central nervous system and probe histone modifications characteristic of active promoters, enhancers and gene bodies (H3K4me3, H3K27ac and H3K36me3) and inactive regions (H3K27me3). These scCUT&Tag profiles were sufficient to determine cell identity and deconvolute regulatory principles such as promoter bivalency, spreading of H3K4me3 and promoter–enhancer connectivity. We also used scCUT&Tag to investigate the single-cell chromatin occupancy of transcription factor OLIG2 and the cohesin complex component RAD21. Our results indicate that analysis of histone modifications and transcription factor occupancy at single-cell resolution provides unique insights into epigenomic landscapes in the central nervous system. An improved method for single-cell analysis of histone modifications is applied to the mouse brain.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper develops single-cell CUT&Tag (scCUT&Tag) by coupling CUT&Tag with droplet-based 10x Genomics scATAC-seq, enabling high-throughput profiling of histone modifications and transcription factor occupancy in complex tissues. Applied to tens of thousands of mouse CNS cells, it resolves major brain cell types and reveals regulatory features such as promoter bivalency, H3K4me3 spreading, and enhancer-promoter connectivity.

Key findings

  • scCUT&Tag generated profiles for 47,340 single cells across histone modifications, with median unique fragments per cell ranging from 98 (H3K36me3) to 453 (H3K27ac); 39.4%–85.6% of fragments fell within narrow peak regions.
  • In a three-cell-line mixture, H3K27me3 scCUT&Tag yielded 4,872 and 3,873 single cells with 597 and 568 unique fragments per cell, respectively, and separated mESC, NIH-3T3, and Oli-neu populations.
  • In mouse brain, scCUT&Tag identified major CNS populations including mature oligodendrocytes, astrocytes, OECs, vascular cells, OPC/COP/NFOL, neurons, and microglia; H3K4me3 breadth increased during oligodendrocyte differentiation, and H3K27me3 was enriched in oligodendrocytes, microglia, and a subset of neurons.
  • TF scCUT&Tag recovered Olig2 and Rad21 binding with median 48 and 240 unique reads per cell, respectively; ABC modeling predicted ~200,000 enhancer-promoter loops, refined to 61,000 with H3K4me3 and ~5,000 with Rad21 on both sides, and Cicero found 14,322 correlated features with score >0.2 in mOLs.

Methods

  • Design: CUT&Tag was combined with droplet-based 10x Genomics single-cell ATAC-seq library preparation; nuclei from mouse CNS or cell lines were barcoded after antibody-directed tagmentation.
  • Sample/data: Mouse Sox10:Cre/RCE brains at P15 and P21–P25 were FACS-sorted into GFP+ and GFP− cells; antibodies targeted H3K4me3, H3K27ac, H3K36me3, H3K27me3, Olig2, and Rad21.
  • Analysis: Cell-feature matrices used 5-kb or 50-kb bins; clustering used LSI, UMAP, SNN, and Leiden in Seurat/Signac; integration used CCA with scRNA-seq; enhancer-promoter predictions used the ABC model and Cicero, with HiChIP validation.

Limitations

  • The paper states that scCUT&Tag currently distinguishes major cell types but fails to uncover subpopulation heterogeneity in an unsupervised manner; oligodendrocyte heterogeneity was only revealed at H3K4me3 level after integration with scRNA-seq.
  • The paper states that TF scCUT&Tag data are sparser than histone scCUT&Tag, and Rad21 binding could not distinguish vascular cells and OPCs, the smallest populations sharing features with larger populations.
  • The paper states that improving unique fragments per cell and introducing multi-omic approaches, such as simultaneous scCUT&Tag and RNA-seq from the same cell, would further enhance applications; evident from the text, TF profiles required annotation assumptions based on correlation with H3K4me3/H3K27ac rather than direct marker-based clustering.

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

Integrity signals

Registry

Crossref · 10.1038/s41587-021-00869-9

Cites

56 of 56 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 22 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Single-cell CUT&Tag data could deconvolute promoter bivalency.

    “These scCUT&Tag profiles were sufficient to determine cell identity and deconvolute regulatory principles such as promoter bivalency, spreading of H3K4me3, and promoter-enhancer connectivity.”
    Quote in the paperSubstantial claim
  • Findingown data

    Single-cell CUT&Tag data for histone marks in the mouse central nervous system could identify cell types.

    “These scCUT&Tag profiles were sufficient to determine cell identity and deconvolute regulatory principles such as promoter bivalency, spreading of H3K4me3, and promoter-enhancer connectivity.”
    Quote in the paperSubstantial claim
  • Findingown data

    Single-cell CUT&Tag data could reveal spreading of H3K4me3.

    “These scCUT&Tag profiles were sufficient to determine cell identity and deconvolute regulatory principles such as promoter bivalency, spreading of H3K4me3, and promoter-enhancer connectivity.”
    Quote in the paperSubstantial claim
  • Findingown data

    Combining CUT&Tag with droplet-based single-cell library preparation yields high-quality single-cell data on chromatin modifications.

    “Here we combine the CUT&Tag technology, developed to measure bulk histone modifications, with droplet-based single-cell library preparation to produce high-quality single-cell data on chromatin modifications.”
    Quote in the paperSubstantial claim
  • Findingown data

    Single-cell CUT&Tag data could reveal promoter-enhancer connectivity.

    “These scCUT&Tag profiles were sufficient to determine cell identity and deconvolute regulatory principles such as promoter bivalency, spreading of H3K4me3, and promoter-enhancer connectivity.”
    Quote in the paperSubstantial claim
  • May combine two claims. 35
  • Classified as probably not a substantial claim. 10

  • May combine two claims.

    Findingown data

    H3K27me3 was associated with genes where active markers were absent, or coincided with H3K4me3.

    “In contrast, H3K27me3 was associated with genes where the other active markers were absent, or it coincided with H3K4me3.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Manual annotation identified mature oligodendrocytes, astrocytes, olfactory ensheathing cells, vascular cells, and oligodendrocyte progenitor spectrum cells in the GFP+ fraction.

    “we manually annotated the populations as mature oligodendrocytes (MOL, Mbp+, Mog+, Cldn11+), astrocytes (AST, Slc1a2+, Rfx4+, Aqp4+), olfactory ensheathing cells (OEC, Alx3+, Alx4+, Frzb+), vascular cells (VAS, Nes+, Tbx18+, Foxf2+) and cells within the spectrum of oligodendrocyte progenitors cells (OPCs), committed OPCs (COPs) and newly formed oligodendrocytes (NFOLs) (OPC, Pdgfra+, Neu4+, Gpr17+”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Bulk and single-cell tracks showed similarity between corresponding populations and lower background in single-cell CUT&Tag.

    “Inspection of genome browser tracks revealed similarity between corresponding populations of bulk and single-cell tracks and lower level of background signal in scCUT&Tag data (Extended Data Figure 6a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Adding BSA did not substantially change tagmentation efficiency or signal distribution in bulk CUT&Tag.

    “We found that addition of BSA reduced nuclei clumping (Extended Data Figure 1b) but did not substantially alter the efficiency of tagmentation nor the signal distribution for bulk Cut&Tag (Extended Data Figure 1c).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Merged pseudo-bulk single-cell CUT&Tag datasets showed distinct profiles for H3K4me3, H3K27ac, H3K36me3 and H3K27me3, indicating specificity.

    “Inspection of merged pseudo-bulk datasets including all cells revealed distinct profiles for the histone modifications highlighting the specificity of the technique (Extended Data Figure 3a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Single-cell CUT&Tag profiles of several histone modifications were obtained for 47340 single cells, with median unique fragments per cell between 98 and 453.

    “Altogether we obtained scCut&Tag profiles of various histone modifications for 47340 single cells with median ranging between 98 (H3K36me3) and 453 (H3K27ac) unique fragments per cell (Figure 1c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Clustering was highly reproducible among biological replicates, and P15 and P25 cells intermingled within clusters.

    “The clustering was highly reproducible among biological replicates and cells originating from P15/P25 age were well intermingled within the clusters (Extended Data Figure 5a–c).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Oligodendrocyte lineage cell states reflected mouse age, with most OPCs from P15 and differentiated oligodendrocytes from P25.

    “The cell states of OLG lineage reflect the mouse age with majority of OPCs originating from P15 and differentiated OLG coming from P25 (Extended Data Figure 5b, c).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Between 39.4% and 85.6% of fragments fell within narrow peak regions, indicating low background.

    “Between 39.4% and 85.6% of fragments fell within narrow peak regions (Figure 1d), indicating a low level of background.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    H3K27me3 single-cell CUT&Tag showed similar or higher specificity, equal or lower unique fragments per cell, but more cells per experiment than iCell8 single-cell CUT&Tag and scChIP-seq.

    “Our scCUT&Tag data showed similar or higher specificity (as measured by fraction of fragments in peaks), equal or lower number of unique fragments per cell, but yielded more cells per experiment compared to the iCell8 scCUT&Tag6 and the latest scChIP-seq method12 (Extended Data Figures 3d-f).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [6] Kaya-Okur, H. S. et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat. Commun. 10, 1930 (2019). source

      Not checked in the free run.

    • cites [12] Grosselin, K. et al. High-throughput single-cell ChIP–seq identifies heterogeneity of chromatin states in breast cancer. Nat. Genet. 51, 1060–1066 (2019). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Fingerprint plots showed single-cell CUT&Tag had higher specificity and better signal-to-noise than scChIP-seq and similar specificity to iCell8 single-cell CUT&Tag.

    “Consistent with this, fingerprint plots showed that scCUT&Tag displayed higher specificity and better signal to noise ratio compared to scChIP-seq12 and similar level of specificity to the iCell8 scCUT&Tag (Extended Data Figure 3g).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [12] Grosselin, K. et al. High-throughput single-cell ChIP–seq identifies heterogeneity of chromatin states in breast cancer. Nat. Genet. 51, 1060–1066 (2019). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Median unique reads per cell were 48 for Olig2 and 240 for Rad21 single-cell CUT&Tag.

    “Nevertheless, we were able to obtain in median 48 and 240 unique reads per cell in Olig2 and Rad21 scCUT&Tag respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Low binder non-OLG cells randomly co-embedded with OEC, astrocyte and vascular clusters defined by H3K27ac, whereas the OLG cluster specifically co-embedded with OLG H3K27ac signal.

    “Interestingly, the low binder non-OLGs cells randomly co-embedded with the populations of OEC, Astrocytes and vascular cell clusters that were defined by H3K27ac, whereas OLG cluster specifically co-embeded with the OLG H3K27ac signal (Extended Data Figure 9e).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The Olig2 dataset separated into two clusters based on depth, and the low-read cluster was annotated as low binders.

    “Olig2 dataset separated into 2 clusters based on depth (number of unique reads per cell), and we annotated the cluster with low number of unique reads as “low binders” (Figure 5a).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    These filtered loops showed substantially higher specificity in HiChIP pileup signal than unfiltered loops and higher overlap between ABC predictions from bulk CUT&Run and single-cell CUT&Tag.

    “These loops showed substantially higher specificity in the HiChIP pileup signal, compared to the original set of unfiltered loops (Figure 6c) and higher overlap between ABC predictions from bulk CUT&Run and scCUT&Tag (Extended Data Figure 10a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CCA integration of active modifications recapitulated the original non-supervised clustering with great precision, and clusters annotated with the same cell type in different datasets occupied the same low-dimensional space.

    “Strikingly, the obtained two-dimensional representation of the data recapitulated the original non-supervised clustering with great precision (Figure 3e) and the clusters annotated with the same cell type in different datasets co-occupied the same low dimensional space (Figure 3e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Olig2 is highly expressed throughout the OLG lineage, whereas OECs and VLMCs do not express Olig2 and only a small portion of astrocytes express Olig2.

    “This finding is consistent with expression of Olig2 throughout the cell types, being highly expressed throughout the OLG lineage, whereas OECs and VLMCs do not express Olig2 and only small portion of astrocytes express Olig2 (Figure 5f).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Co-embedding H3K4me3 single-cell CUT&Tag and single-cell RNA-seq using CCA showed good integration while RNA-seq clustering was retained.

    “For this purpose, we co-embedded the H3K4me3 scCUT&Tag and scRNA-seq using CCA, which showed good integration of the techniques, while the scRNA-seq clustering was retained (Figure 3b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    GO terms such as astrocyte differentiation, myelination, regulation of myelination, vasculogenesis migration, glial development, neuron development and microglial activation were specifically enriched in respective clusters.

    “found GO terms such as astrocyte differentiation and activation (AST), myelination (OLG), regulation of myelination (OEC), cell migration involved in vasculogenesis (VAS), glial cell development (OPCs), neuron development, neuron maturation and axonogenesis (NEU) and microglial cell activation involved in immune response (MGL) (Extended Data Figure 8) to be specifically enriched in respective”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    An increase in H3K4me3 breadth during oligodendrocyte lineage differentiation was observed, indicating that spreading of this modification can be used to delineate lineage progression.

    “We also observed an increase in H3K4me3 breadth during the process of differentiation in the OLG lineage at unprecedented resolution, indicating that spreading of this modification can be used to delineate lineage progression.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Analysis of histone modifications at single-cell level allows identification of distinct neural cell types, with H3K4me3, H3K27ac and H3K27me3 particularly useful.

    “We show that analysis of histone modifications at a single-cell level allows identification of distinct neural cell types, with the active marks H3K4me3 and H3K27ac and the repressive mark H3k27me3 being particularly useful for this purpose.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Single-cell CUT&Tag can distinguish major cell types but fails to uncover heterogeneity of subpopulations in an unsupervised manner.

    “In the current state, scCUT&Tag is able to distinguish the major cell types but fails to uncover heterogeneity of the subpopulations in an unsupervised manner.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Olig2 preferentially binds oligodendrocyte lineage-specific enhancers and promoters and binds only weakly to astrocyte-specific gene regulatory regions.

    “While Olig2 preferentially binds oligodendrocyte lineage specific enhancers and promoters and binds only weakly to astrocyte-specific gene regulatory regions, Rad21, a ubiquitously expressed genome architecture factor, binds cell type–specific promoters and enhancers.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Cicero predictions modestly overlapped with ABC model predictions, and predicted loop length was substantially different between Cicero and the ABC model.

    “Cicero predictions modestly overlapped with ABC model predictions (Extended Data Figure 10b) and the length of the predicted loops was substantially different between cicero and ABC model (Extended Data Figure 10d).”
    Quote in the paperNot a substantial claim

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

    Findingnone given

    Breeding males carrying a hemizygous Cre allele and the reporter allele with non-Cre carrier females causes early Cre activation throughout the animal, so this breeding scheme was avoided.

    “Breeding with males containing a hemizygous Cre allele in combination with the reporter allele to non-Cre carrier females resulted in early Cre activation in the whole animal and consequently avoided.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Single-cell CUT&Tag allowed deconvolution of potential H3K4me3/H3K27me3 bivalency and revealed a close epigenetic relationship between OPCs and astrocytes.

    “scCUT&Tag allowed the deconvolution of potential H3K4me3/H3K27me3 bivalency, but also revealed a close epigenetic relationship between OPCs and astrocytes.”
    Quote in the paperSubstantial claim

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

    Findingown data

    Additional motifs were identified using MEME from the merged pseudobulk profile of Olig2 scCUT&Tag.

    “Additional motifs identified using MEME from the merged pseudobulk profile of Olig2 scCUT&Tag.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Both prediction methods identified known Sox10 enhancers and connected them to the Sox10 promoter.

    “Nevertheless, both predictions methods successfully identified known Sox10 enhancers 40 and connected them to the Sox10 promoter (Figure 6e).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [40] Werner, T., Hammer, A., Wahlbuhl, M., Bösl, M. R. & Wegner, M. Multiple conserved regulatory elements with overlapping functions determine Sox10 expression in mouse embryogenesis. Nucleic Acids Res. 35, 6526–6538 (2007). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    The astrocyte population detected in the GFP+ fraction is likely transient and most probably derived from ventral regions.

    “Interestingly, we detected a major, likely transient population of astrocytes, in GFP+ fraction of the Sox10:Cre/RCE mouse (Extended Data Figure 2a, 2c), most probably derived from ventral regions22–24.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [22] Huang, W. et al. Novel NG2-CreERT2 knock-in mice demonstrate heterogeneous differentiation potential of NG2 glia during development. Glia 62, 896–913 (2014). source

      Not checked in the free run.

    • cites [23] Zhu, X. et al. Age-dependent fate and lineage restriction of single NG2 cells. Development 138, 745–753 (2011). source

      Not checked in the free run.

    • cites [24] Zhu, X., Bergles, D. E. & Nishiyama, A. NG2 cells generate both oligodendrocytes and gray matter astrocytes. Development 135, 145–157 (2008). source

      Not checked in the free run.

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

    Premiseown data

    CTCF and Cohesin cooperate.

    “We found the motif of chromatin architecture factor, CTCF as the highest enriched in the Rad21 dataset (Figure 5g), which is consistent with the cooperativity between Ctcf and Cohesin32.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] Li, Y. et al. The structural basis for cohesin–CTCF-anchored loops. Nature 578, 472–476 (2020). source

      Not checked in the free run.

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

    Premisecitation

    Disruption of H3K27me3 impairs OPC differentiation to MOL and triggers a switch towards an astrocytic fate.

    “Consistently, it was recently reported that disruption of H3K27me3 impairs OPC differentiation to MOL, and triggers switch towards an astrocytic faith29.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [29] Wang, J. et al. EED-mediated histone methylation is critical for CNS myelination and remyelination by inhibiting WNT, BMP, and senescence pathways. Sci. Adv. 6, eaaz6477 (2020). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Signal heterogeneity is unlikely caused by differential tagmentation efficiency between cell types and is more likely caused by differential modification levels between cell types.

    “Therefore, it is unlikely that the signal heterogeneity is caused by differential tagmentation efficiency between cell types but rather by differential levels of modifications between the cell types.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The number of unique reads per cell reflects the absolute amount of a histone modification in single cells.

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

    Premisecitation

    Olig2 physically interacts and cooperates with Sox8 and Sox10.

    “Interestingly, we also found a motif similar to the motif of transcription factors from the Sox family (ACARWR, Extended Data Figure 9f), which is consistent with physical interaction and cooperativity between Olig2 and members of Sox family of transcription factors (Sox8, Sox10)35.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [35] Wißmüller, S., Kosian, T., Wolf, M., Finzsch, M. & Wegner, M. The high-mobility-group domain of Sox proteins interacts with DNA-binding domains of many transcription factors. Nucleic Acids Res. 34, 1735–1744 (2006). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The OLG population in Olig2 single-cell CUT&Tag is likely composed of mature OLG and OPCs, which appear to form a subpopulation within the OLG cluster.

    “The OLG population in Olig2 scCUT&Tag is likely composed of both mature OLG and OPCs, which appear to form a subpopulation within the OLG cluster (Extended Data Figure 9a).”
    Quote in the paperNot a substantial claim
    • The authors state this as an assumption: Olig2 and Rad21 binding in specific cell types correlates with enhancer and promoter activity.

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

    Premisecitation

    The breadth of H3K4me3 has been linked to cell identity, gene expression and transcriptional consistency across cell types.

    “The breadth of the H3K4me3 mark has been previously linked to cell identity, gene expression and transcriptional consistency across a variety of cell types30.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [30] Benayoun, B. A. et al. H3K4me3 breadth is linked to cell identity and transcriptional consistency. Cell 158, 673–688 (2014). source

      Not checked in the free run.

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

    Study design and conductnone given

    The battery of screened infective agents met the standard health profile established in Karolinska Institutet animal housing facilities.

    “The battery of screened infective agents met the standard health profile established in Karolinska Institutet animal housing facilities.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    HiChIP was performed in three biological replicates with at least 1 million cells as input.

    “HiChIP was performed in three biological replicates with at least 1 milion cells used as input.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    scRNA-seq was performed in two biological replicates.

    “Then, cells were counted, and ~7000 cells processed using chromium single cell 3’ reagent kit (10x Genomics) according to manufacturer’s instructions in two biological replicates.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All animals were free from the most common mouse viral pathogens, ectoparasites, endoparasites, and mouse bacteria pathogens harbored in research animals.

    “All animals were free from the most common mouse viral pathogens, ectoparasites, endoparasites, and mouse bacteria pathogens harbored in research animals.”
    Quote in the paperNot a substantial claim
39 of 39

  • The authors state this as an assumption.

    Olig2 and Rad21 binding in specific cell types correlates with enhancer and promoter activity.

    “we based cluster annotation on the assumption that Olig2/Rad21 binding in specific cell types is correlated with enhancer/promoter activity.”

  • The paper relies on this without support.

    The number of unique reads per cell reflects the absolute amount of a histone modification in single cells.

    “We used the number of unique reads per cell as a proxy for absolute amount of the histone modification in single cells.”
2 of 2

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • scCUT&Tag integrated CUT&Tag with 10x Genomics scATAC-seq protocol, enabling investigation of histone modifications at single-cell resolution.

    “single-cell CUT&Tag (scCUT&Tag) integrated CUT&Tag with 10× Genomics scATAC-seq protocol, enabling the investigation of histone modifications at single-cell resolution [120].”

    From Single-cell sequencing to multi-omics: technologies and applications (2024) · cites this paper as [120]

  • scCUT&Tag and scCUT&Tag2for1 use antibody-guided tagmentation to map active and silenced regulatory elements across the genome by targeting RNA polymerase II and Polycomb complexes, respectively.

    “This method has been modified further by other groups to profile histone modifications at the single-cell level: methods such as scCUT&Tag48 and scCUT&Tag2for149 use tagmentation directed by antibodies to profile active and silenced regulatory elements genome-wide by targeting domains bound by, respectively, RNA polymerase II and Polycomb repressive complexes.”

    From The technological landscape and applications of single-cell multi-omics (2023) · cites this paper as [48]

  • Integration and co-embedding of spatial CUT&Tag and scCUT&Tag data showed that the epigenetic states in the study data for H3K27me3, H3K27ac and H3K4me3 agreed with the corresponding projection in scCUT&Tag.

    “Integration and co-embedding of spatial CUT&Tag and scCUT&Tag data36,37 (Fig. 3d,e and Extended Data Fig. 7a) showed that the epigenetic states in our data for H3K27me3, H3K27ac and H3K4me3 agreed with the corresponding projection in scCUT&Tag.”

    From Spatial epigenome–transcriptome co-profiling of mammalian tissues (2023) · cites this paper as [36]

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.

  • Conflicting ABC-score thresholds

    Major

    The ABC model methods specify a threshold of 0.2 for downstream analyses, whereas the Statistics section calls predictions above 0.02 significant. These thresholds would select different sets of predicted loops.

  • Bulk assay used for Figure 10a is inconsistent

    Minor

    The Figure 10a caption identifies the bulk comparison as CUT&Tag, while the Results identify the comparison in Figure 10a and b as bulk Cut&Run.

  • Animal age range excludes the reported P15 cohort

    Minor

    The Animals section says animals were sacrificed only at P4-P6 or P21-25, but the Results report sorting brain cells at P15.

3 of 3

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Related

Authors
Bartosovic, M., Kabbe, M., & Castelo-Branco, G.
Affiliations
Karolinska Institutet (SE)
ORCID
Venue
Nature Biotechnology
Publisher
Springer Science and Business Media LLC
Published
2021-04-12
Volume
39
Issue
7
Pages
825-835
ISSN
  • 0733-222X (ISSN-L)
  • 1087-0156
  • 1546-1696
DOI
10.1038/s41587-021-00869-9
PMC
PMC7611252
OpenAlex
W3156830838
Semantic Scholar
e3838c1afa483c16cb19e88f8243c4e159ab6149
Cited by
483
References
54
Citations by year
2026: 96 · 2025: 94 · 2024: 106 · 2023: 93 · 2022: 63 · 2021: 30 · 2019: 1
FWCI
23.47
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Biotechnology (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
Keywords
histone modifications, chromatin accessibility, transcription factor occupancy, Olig2, RAD21, cohesin, H3K4me3, H3K27ac, H3K36me3, H3K27me3, central nervous system, mice, epigenomics
MeSH terms
Animals, Brain, Gene Expression Regulation, Histones, Humans, Transcription Factors, Cluster Analysis, Mice, Single-Cell Analysis

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 2026-10-07.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-07.

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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