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Single-cell CUT&Tag analysis of chromatin modifications in differentiation and tumor progression


Methods for quantifying gene expression1 and chromatin accessibility2 in single cells are well established, but single-cell analysis of chromatin regions with specific histone modifications has been technically challenging. In this study, we adapted the CUT&Tag method3 to scalable nanowell and droplet-based single-cell platforms to profile chromatin landscapes in single cells (scCUT&Tag) from complex tissues and during the differentiation of human embryonic stem cells. We focused on profiling polycomb group (PcG) silenced regions marked by histone H3 Lys27 trimethylation (H3K27me3) in single cells as an orthogonal approach to chromatin accessibility for identifying cell states. We show that scCUT&Tag profiling of H3K27me3 distinguishes cell types in human blood and allows the generation of cell-type-specific PcG landscapes from heterogeneous tissues. Furthermore, we used scCUT&Tag to profile H3K27me3 in a patient with a brain tumor before and after treatment, identifying cell types in the tumor microenvironment and heterogeneity in PcG activity in the primary sample and after treatment.

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Fig. 1: scCUT&Tag resolves distinct cell types and maps repressive chromatin domains in early hESC development.
Fig. 2: scCUT&Tag for H3K27me3 readily identifies major subtypes in PBMCs.
Fig. 3: scCUT&Tag data for H3K27me3 for a human glioblastoma primary and relapse sample demonstrate heterogeneity in PcG distribution within tumor cell clusters and cluster enrichment after treatment.

Data availability

Sequencing data are deposited in the Gene Expression Omnibus with accession code GSE157910. There are no restrictions on data use.

Code availability

Code used in this study can be found on GitHub at


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We thank E. Holland and members of the Holland lab for providing shared space for experimental work, the Fred Hutchinson Genomics Shared Resource for DNA sequencing and microfluidics services and BioRender for helping create figures. We thank T. Bryson, C. Codomo, J. Henikoff, M. Meers, D. Janssens, M. Setty, J. Thakur and other members of the Henikoff lab for helpful suggestions and discussions. We also thank the Koeplin Family Foundation and the Nancy and Buster Alvord Endowment, as well as A. Schantz for administrative support and L. Keene, A. Keen and K. Kern for technical support with autopsy specimen collection. This work was supported by the Howard Hughes Medical Institute (to S.H.); grants R01 HG010492 (to S.H.), R01 GM108699 (to K.A.) and K08 CA245037 (to P.J.C.) from the National Institutes of Health; an HCA Seed Network grant from the Chan-Zuckerberg Initiative (to S.H., A.P.P, S.N.F, R.G., K.A. and Y.Z.); a Burroughs Wellcome Career Award for Medical Scientists (to A.P.P.); and an American Cancer Society Mentored Scholar Award (to S.N.F). The Scientific Computing Infrastructure at the Fred Hutchinson Cancer Research Center is funded by ORIP grant S10OD028685.

Author information




S.J.W., S.N.F., A.B.M., H.K-O., A.H.F., S.N.E., and J.F.S. processed samples and performed experiments. S.J.W., S.N.F., Y.Z., R.G. and A.P.P. performed and/or provided input on data processing and analysis. K.C., P.J.C. and C.D.K. provided access to tissue samples and assisted with processing. S.J.W., S.N.F., K.A., S.H. and A.P.P. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Steven Henikoff or Anoop P. Patel.

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Competing interests

S.N.F. has received research support from Lyell Immunopharma. R.G. has received consulting income from Juno Therapeutics, Takeda, INFOTECHSoft, Celgene and Merck; has received research support from Janssen Pharmaceuticals and Juno Therapeutics; and declares ownership in CellSpace Biosciences. H.S.K. and S.H. have filed patent applications related to this work. A.P.P. declares ownership in Sygnomics.

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Supplementary Figs. 1–8 and Table 1.

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Wu, S.J., Furlan, S.N., Mihalas, A.B. et al. Single-cell CUT&Tag analysis of chromatin modifications in differentiation and tumor progression. Nat Biotechnol 39, 819–824 (2021).

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