Abstract
The epigenome of a cell is tightly correlated with gene transcription, which controls cell identity and diverse biological activities. Recent advances in spatial technologies have improved our understanding of tissue heterogeneity by analyzing transcriptomics or epigenomics with spatial information preserved, but have been mainly restricted to one molecular layer at a time. Here we present procedures for two spatially resolved sequencing methods, spatial-ATAC-RNA-seq and spatial-CUT&Tag-RNA-seq, that co-profile transcriptome and epigenome genome wide. In both methods, transcriptomic readouts are generated through tissue fixation, permeabilization and in situ reverse transcription. In spatial-ATAC-RNA-seq, Tn5 transposase is used to probe accessible chromatin, and in spatial-CUT&Tag-RNA-seq, the tissue is incubated with primary antibodies that target histone modifications, followed by Protein A-fused Tn5-induced tagmentation. Both methods leverage a microfluidic device that delivers two sets of oligonucleotide barcodes to generate a two-dimensional mosaic of tissue pixels at near single-cell resolution. A spatial-ATAC-RNA-seq or spatial-CUT&Tag-RNA-seq library can be generated in 3–5 d, allowing researchers to simultaneously investigate the transcriptomic landscape and epigenomic landscape of an intact tissue section. This protocol is an extension of our previous spatially resolved epigenome sequencing protocol and provides opportunities in multimodal profiling.
Key points
Spatially resolved concurrent profiling of both transcriptome and epigenome is essential for studying spatio-temporal regulation of gene expression, but the technical solutions permitting such analyses remain limited.
This protocol describes spatial-ATAC-RNA-seq and spatial-CUT&Tag-RNA-seq, which profile genome-wide transcription jointly with open chromatin and histone modifications, respectively, on a tissue section.
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Fig. 1: Overview of the procedures for spatial-ATAC-RNA-seq and spatial-CUT&Tag-RNA-seq.
Fig. 2: Library structure and oligonucleotide primer sequence design.
Fig. 3: Procedures for tissue fixation, permeabilization and Tn5 or pA-Tn5 tagmentation.
Fig. 4: Workflow of deterministic barcoding in tissue.
Fig. 5: Anticipated results of library visualization.
Fig. 6: Bioinformatics pipelines for analyzing spatially resolved epigenome-transcriptome data.
Fig. 7: Spatial-ATAC-RNA-seq and spatial-CUT&Tag-RNA-seq data quality control.
Fig. 8: Evaluation and analysis of a spatial-ATAC-RNA-seq dataset.
Data availability
Original data that are used to generate metrics plots in Figs. 7 and 8 are available in Source Data. Raw data for the illustrative results shown in this protocol are available in Zhang et al.26. Source data are provided with this paper.
Code availability
Codes for processing data of the associated publication26 are available at https://github.com/di-0579/Spatial_epigenome-transcriptome_co-sequencing.
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Acknowledgements
We thank the Yale Center for Research Computing for guidance and use of the research computing infrastructure. The molds for microfluidic devices were fabricated at the Yale University School of Engineering and Applied Science Nanofabrication Center. Next-generation sequencing was conducted at the Yale Center for Genome Analysis, as well as at the Yale Stem Cell Center Genomics Core Facility, which was supported by the Connecticut Regenerative Medicine Research Fund and the Li Ka Shing Foundation. A service provided by the Genomics Core of Yale Cooperative Center of Excellence in Hematology (U54DK106857) was used. This research was supported by Packard Fellowship for Science and Engineering (to R.F.), Yale Stem Cell Center Chen Innovation Award (to R.F.), and grants from the US National Institutes of Health (U54AG076043, U54AG079759, UG3CA257393, UH3CA257393, R01CA245313, RF1MH128876, U54CA274509, U54CA268083 and U01CA294514 to R.F.). Illustrations were created with BioRender.com.
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Authors and Affiliations
Department of Biomedical Engineering, Yale University, New Haven, CT, USA
Haikuo Li, Shuozhen Bao, Negin Farzad, Xiaoyu Qin, Anthony A. Fung, Di Zhang, Zhiliang Bai & Rong Fan
Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
Bo Tao & Rong Fan
Yale Stem Cell Center and Yale Cancer Center, Yale University School of Medicine, New Haven, CT, USA
Rong Fan
Yale Center for Research on Aging (Y-Age), Yale University School of Medicine, New Haven, CT, USA
Rong Fan
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Haikuo Li
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Contributions
H.L., N.F. and R.F. conceived, coordinated and designed the study. H.L., S.B., X.Q. and B.T. wrote the manuscript. H.L., S.B., A.A.F., D.Z., Z.B. and B.T. analyzed data and created figures. R.F. supervised the project and revised the manuscript. All authors read and approved the final manuscript.
Corresponding author
Correspondence to Rong Fan.
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Competing interests
R.F. is scientific founder of and advisor to IsoPlexis, Singleron Biotechnologies and AtlasXomics. The Yale University Provost’s Office reviewed and managed the interests of R.F. in accordance with the University’s conflict of interest policies. The other authors declare no competing interests.
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Additional information
Key references
Zhang, D. et al. Spatial epigenome–transcriptome co-profiling of mammalian tissues. Nature 616, 113–122 (2023): https://doi.org/10.1038/s41586-023-05795-1
Farzad, N. et al. Spatially resolved epigenome sequencing via Tn5 transposition and deterministic DNA barcoding in tissue. Nat. Protoc. 19, 3389–3425 (2024): https://doi.org/10.1038/s41596-024-01013-y
This protocol is an extension to: Nat. Protoc. 19, 3389–3425 (2024): https://doi.org/10.1038/s41596-024-01013-y
Extended data
Extended Data Fig. 1 Anticipated results of spatial-ATAC-seq library visualization.
TapeStation D5000 electropherogram (High-Sensitivity) showing fragment distribution of a spatial-ATAC-seq library generated from a human brain sample. The table on the right panel indicates the concentration, molarity and fractions of fragments in the select region (200-5,000 bp).
Supplementary information
Reporting Summary
Supplementary Table 1
Oligo sequence information.
Supplementary Data 1
Microfluidic CAD designs.
Supplementary Video 1
Video illustration for attaching PDMS reservoirs on the tissue.
Supplementary Video 2
Video illustration for ATAC-seq and CUT&Tag related procedures.
Supplementary Video 3
Video illustration for attaching PDMS chips on the tissue.
Supplementary Video 4
Video illustration for streptavidin-bead affinity pulldown.
Supplementary Video 5
Video illustration for PDMS associated equipment preparation.
Source data
Source Data Figs. 7 and 8
Original data used to generate metrics plots in Figs. 7 and 8.
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Li, H., Bao, S., Farzad, N. et al. Spatially resolved genome-wide joint profiling of epigenome and transcriptome with spatial-ATAC-RNA-seq and spatial-CUT&Tag-RNA-seq. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01145-9
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Received:29 June 2024
Accepted:15 November 2024
Published:21 March 2025
DOI:https://doi.org/10.1038/s41596-025-01145-9
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