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Nano-CUT&Tag for multimodal chromatin profiling at single-cell resolution

Nature Protocols volume 19, pages 791–830 (2024)Cite this article

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Abstract

The ability to comprehensively analyze the chromatin state with single-cell resolution is crucial for understanding gene regulatory principles in heterogenous tissues or during development. Recently, we developed a nanobody-based single-cell CUT&Tag (nano-CT) protocol to simultaneously profile three epigenetic modalities—two histone marks and open chromatin state—from the same single cell. Nano-CT implements a new set of secondary nanobody-Tn5 fusion proteins to direct barcoded tagmentation by Tn5 transposase to genomic targets labeled by primary antibodies raised in different species. Such nanobody-Tn5 fusion proteins are currently not commercially available, and their in-house production and purification can be completed in 3–4 d by following our detailed protocol. The single-cell indexing in nano-CT is performed on a commercially available platform, making it widely accessible to the community. In comparison to other multimodal methods, nano-CT stands out in data complexity, low sample requirements and the flexibility to choose two of the three modalities. In addition, nano-CT works efficiently with fresh brain samples, generating multimodal epigenomic profiles for thousands of brain cells at single-cell resolution. The nano-CT protocol can be completed in just 3 d by users with basic skills in standard molecular biology and bioinformatics, although previous experience with single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) is beneficial for more in-depth data analysis. As a multimodal assay, nano-CT holds immense potential to reveal interactions of various chromatin modalities, to explore epigenetic heterogeneity and to increase our understanding of the role and interplay that chromatin dynamics has in cellular development.

Key points

  • This protocol involves profiling gene regulatory dynamics in complex tissues at the single-cell level. This is achieved by generating two nanobody-Tn5 fusion proteins to recognize primary antibodies against widespread histone modifications.

  • The nanobody-Tn5 fusions are combined with ATAC-seq to simultaneously profile three epigenetic modalities from the same single cell, thousands of cells at the same time. A bioinformatic pipeline, Nanoscope, for seamless analysis of nano-CT datasets is also described.

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Fig. 1: Schematics overview of the entire procedure for multimodal single-cell Nano-CUT&Tag (nano-CT).
Fig. 2: Schematic overview of the procedure covering nanobody-Tn5 fusion protein expression and purification.
Fig. 3: Example purification SEC chromatograms and SDS-PAGE analysis of fractions and final batches.
Fig. 4: QC for nano-CT library product size distribution.
Fig. 5: Overview of the steps performed in the Nanoscope pipeline and nano-CT data analysis.
Fig. 6: QC for nano-CT data.
Fig. 7: Multimodal nano-CT.
Fig. 8: ATAC, H3K27ac and H3K27me3 at loci containing mature oligodendrocyte and inhibitory neuronal marker genes.

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Data availability

Raw nano-CT data were deposited in Gene Expression Omnibus under accession GSE198467. Source data are provided with this paper.

Code availability

All code, scripts, analysis pipeline and instructions for reproducibility can be found on Github at https://github.com/bartosovic-lab/nanoscope, and the analysis vignette can be found at https://fansalon.github.io/vignette_single-cell-nano-CT.html.

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Acknowledgements

We thank T. Jimenez-Beristain for writing laboratory animal ethics permit 1995_2019 and assistance with animal experiments; L. Kirby, M. Meijer, C. Zheng and P. Kukanja for assistance with animal experiments; and the staff at Comparative Medicine-Biomedicum. We thank M. Leboeuf and E. Hedlund´s group for valuable suggestions with figures. We acknowledge support from the National Genomics Infrastructure in Stockholm funded by the Science for Life Laboratory, the Knut and Alice Wallenberg Foundation and the Swedish Research Council. Computation/data handling was enabled by resources provided by the Swedish National Infrastructure for Computing (SNIC) at the Uppsala Multidisciplinary Center for Advanced Computational Science, partially funded by the Swedish Research Council through grant agreement number 2022-06725. We thank Abcam for providing a set of histone modification antibodies for CUT&Tag testing. M.B. was funded by the Vinnova Seal of Excellence Marie-Sklodowska Curie Actions grant RNA-centric view on Oligodendrocyte lineage development (RODent). Work in the research group of G.C.-B. was supported by the Swedish Research Council (grant no. 2019-01360), the European Union (Horizon 2020 Research and Innovation Programme/European Research Council Consolidator Grant EPIScOPE, grant agreement number 681893), the Swedish Cancer Society (Cancerfonden; 190394 Pj), the Knut and Alice Wallenberg Foundation (grants 2019-0107 and 2019-0089), the Swedish Society for Medical Research (SSMF, grant JUB2019), the Göran Gustafsson Foundation for Research in Natural Sciences and Medicine, the Ming Wai Lau Centre for Reparative Medicine and the Karolinska Institutet. Work in M.B.’s laboratory is supported by the Swedish Research Council (grant no. 2021-01476), Carl Tryggers Stiftelse (CTS22:2079), Jaensson’s Stiftelse, Ole Engkvist Stiftelse – Swedish foundation’s starting grant and Cancerfonden Junior Investigator Award (23 0758 JIA). F.A. was supported by the European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS) in the scope of the Postdoctoral Research Fellowship. All experimental procedures on animals were performed by following the European directive 2010/63/EU, local Swedish directive L150/SJVFS/2019:9, Saknr L150 and Karolinska Institutet complementary guidelines for the procurement and use of laboratory animals, Dnr. 1937/03–640. The procedures described were approved by the local committee for ethical experiments on laboratory animals in Sweden (Stockholms Norra Djurförsöksetiska nämnd), license number 144/16, 1995_2019 and 7029/2020.

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

    José Ramón Bárcenas-Walls & Marek Bartošovič

  2. Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

    Federico Ansaloni, Bastien Hervé & Gonçalo Castelo-Branco

  3. Protein Science Facility, Department of Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

    Emilia Strandback & Tomas Nyman

  4. Ming Wai Lau Centre for Reparative Medicine, Stockholm node, Karolinska Institutet, Stockholm, Sweden

    Gonçalo Castelo-Branco

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Contributions

M.B. and G.C.-B. designed and developed the nano-CT protocol. M.B. performed the experiments. M.B., F.A. and B.H. performed bioinformatic data analysis and developed the Nanoscope pipeline and vignette. E.S. and T.N. performed the purification of nanobody-Tn5 recombinant protein and wrote the part of the manuscript describing recombinant protein expression and purification. J.R.B.-W. and M.B. wrote the manuscript and prepared the figures.

Corresponding author

Correspondence to Marek Bartošovič.

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Competing interests

G.C.-B. and M.B. have filed a patent application on the basis of this work (European patent application number EP22160860.7).

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Nature Protocols thanks Leif Ludwig and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Bartosovic, M. & Castelo-Branco, G. Nat. Biotechnol. 41, 794–805 (2023): https://doi.org/10.1038/s41587-022-01535-4

Extended data

Extended Data Fig. 1 Workflows for activity assessment of in-house-produced Tn5.

a, Supplementary Method for assessing Tn5 activity on the basis of inverse qPCR detection. Step numbers corresponding to the Supplementary Method A numeration are shown to the right. b, Titration of in-house-produced Tn5 (ME-B) from different batches by qPCR. By assessing the activity of Tn5 assembly batches with respect to fragmenting a known template, we can obtain information about the volume to be used for the tagmentation of LA products from nano-CT library preparation (Step 108 from the Procedure). c, Supplementary Method for unimodal nano-CT in bulk (without single-cell barcoding) to perform titration with Tn5 (ME-B) and use this value for single-cell nano-CT. Step numbers corresponding to the Supplementary Method B numeration are shown to the right. d, Bioanalyzer traces performing nano-CT in bulk, showing different ratios of Tn5 (ME-B) and different size distributions of the final nano-CT libraries. Ct, cycle threshold.

Extended Data Fig. 2 Nano-CT library structure.

a and b, Full amplification sequence with Illumina P5 (a) and P7 (b) sequences for the nano-CT library. Note that both a and b correspond to the same nano-CT library molecule and are divided by genomic insert sequences. The custom primers required are for Read 1 and for Index 2 (shown in purple). Importantly, the ‘Modality barcode (8 nt)’ will contain the sequence from oligo adapters used during transposome complex loading (barcodes A, B and C in Steps 28–32). Library sequences will be demultiplexed by modalities by the Nanoscope pipeline. c, Schematic representation of the nano-CT library structure.

Supplementary information

Source data

Source Data Fig. 3

Unprocessed Coomassie-stained SDS-PAGE gels loaded with 10 µl of each selected fraction from psfaMsTn5 (c) and psfaRbTn5 (d) purifications.

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Bárcenas-Walls, J.R., Ansaloni, F., Hervé, B. et al. Nano-CUT&Tag for multimodal chromatin profiling at single-cell resolution. Nat Protoc 19, 791–830 (2024). https://doi.org/10.1038/s41596-023-00932-6

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