Single-cell ultra-high-throughput multiplexed chromatin accessibility and gene expression sequencing (SUM-seq)

The described computational pipeline for processing SUM-seq sequencing data is available at GitHub via https://git.embl.de/grp-zaugg/SUMseq.

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Author notes

1. Umut Yildiz

   Present address: Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland

2. These authors contributed equally: Umut Yildiz, Sara Lobato-Moreno.

Authors and Affiliations

1. European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany

   Umut Yildiz, Sara Lobato-Moreno, Annique Claringbould, Víctor Campos-Fornés, Karin D. Prummel, Judith B. Zaugg, Kyung Min Noh & Mikael Marttinen

2. Faculty of Biosciences, Collaboration for Joint PhD Degree between EMBL and Heidelberg University, Heidelberg, Germany

   Umut Yildiz, Sara Lobato-Moreno & Víctor Campos-Fornés

3. European Molecular Biology Laboratory, Molecular Systems Biology Unit, Heidelberg, Germany

   Sara Lobato-Moreno, Annique Claringbould, Hanke Gwendolyn Bauersachs, Nila H. Servaas, Evi P. Vlachou, Christian Arnold, Karin D. Prummel, Judith B. Zaugg & Mikael Marttinen

4. Department of Internal Medicine, Erasmus Medical Centre Rotterdam, Rotterdam, the Netherlands

   Annique Claringbould

5. Department of Biomedicine, Basel University, University Hospital Basel, Basel, Switzerland

   Hanke Gwendolyn Bauersachs & Judith B. Zaugg

6. Molecular Medicine Partnership Unit, Heidelberg, Germany

   Judith B. Zaugg

7. Department of Biomedicine, Aarhus University, Aarhus, Denmark

   Kyung Min Noh

8. Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland

   Mikael Marttinen

Contributions

M.M., U.Y. and S.L.-M. designed and developed the core experimental protocol; H.G.B., K.D.P., N.H.S. and V.C.-F. assisted in the development of the experimental protocol; C.A., E.P.V., A.C. and M.M. designed and developed the computational pipeline; U.Y. and M.M. wrote the manuscript with input from all authors; U.Y., S.L.-M. and M.M. produced the figures for the manuscript; M.M., U.Y., K.M.N. and J.B.Z. supervised the project.

Corresponding authors

Correspondence to Umut Yildiz, Judith B. Zaugg, Kyung Min Noh or Mikael Marttinen.

Competing interests

The authors declare no competing interests.

Peer review information

Nature Protocols thanks Marek Bartosovic, Pascal Hunold and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Key reference

Lobato-Moreno, S. et al. Nat. Methods 22, 1213–1225 (2025): https://doi.org/10.1038/s41592-025-02700-8

Extended Data Fig. 1 Comparison of multimodal methods for profiling of chromatin accessibility and gene expression from the same nucleus.

A common step in all strategies is the dissociation of the tissue/sample to obtain a single cell suspension, and the tagmentation of open chromatin following nuclei isolation (1). The commercial 10x Epi multiome workflow continues with the encapsulation of single nuclei into individual droplets, and barcoding of open chromatin and nuclear mRNA, omitting the possibility to pool multiple samples and limiting the throughput (a). While ligation-based combinatorial indexing strategies (b) offer higher throughput and, in principle, allow multiplexing, the capability is constrained by the number of first barcodes utilized. Moreover, multiplexing is experimentally challenging, as barcodes are appended after the in situ chromatin tagmentation and reverse transcription steps. Lastly, the ligation-based barcoding compromises the data complexity and quality. Combinatorial microfluidic indexing, as used in SUM-seq (c) enables a one-step multiplexing option, resulting in increased throughput and high data complexity. Created with BioRender.com.

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