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A plate-based single-cell ATAC-seq workflow for fast and robust profiling of chromatin accessibility

Abstract

Profiling chromatin accessibility at the single-cell level provides critical information about cell type composition and cell-to-cell variation within a complex tissue. Emerging techniques for the interrogation of chromatin accessibility in individual cells allow investigation of the fundamental mechanisms that lead to the variability of different cells. This protocol describes a fast and robust method for single-cell chromatin accessibility profiling based on the assay for transposase-accessible chromatin using sequencing (ATAC-seq). The method combines up-front bulk Tn5 tagging of chromatin with flow cytometry to isolate single nuclei or cells. Reagents required to generate sequencing libraries are added to the same well in the plate where cells are sorted. The protocol described here generates data of high complexity and excellent signal-to-noise ratio and can be combined with index sorting for in-depth characterization of cell types. The whole experimental procedure can be finished within 1 or 2 d with a throughput of hundreds to thousands of nuclei, and the data can be processed by the provided computational pipeline. The execution of the protocol only requires basic techniques and equipment in a molecular biology laboratory with flow cytometry support.

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Fig. 1: Workflow of the protocol.
Fig. 2: Removal of the supernatant.
Fig. 3: Single-nucleus/cell isolation using FACS.
Fig. 4: DNA purification after well barcode addition.
Fig. 5: Typical scATAC-seq library profiles.
Fig. 6: Overview of the Snakemake pipeline is used to process scATAC-seq data.
Fig. 7: Typical results from the data processing pipeline.
Fig. 8: Genome browser view of reads pileup from aggregated scATAC-seq data.
Fig. 9: Comparison of K562 scATAC-seq data obtained using different DNA stains.
Fig. 10: Comparison of PBMC scATAC-seq data from the 10x Genomics and the plate-based methods.

Data availability

The scATAC-seq data from mouse splenocytes, mouse embryonic stem cells and human K562 cells are deposited in ArrayExpress, accession number E-MTAB-6714. The bulk ATAC-seq data from the ImmGen project are available in the European Nucleotide Archive (ENA), study accession number PRJNA392905. The data from K562 cells stained with different DNA staining methods and human PBMCs are deposited in the Genome Sequence Archive in BIG Data Center, under accession number PRJCA004353.

Code availability

The code used to process the data and the instructions to run the code are available in the Zenodo repository under https://doi.org/10.5281/zenodo.4734142 (ref. 58) and in the GitHub repository at https://github.com/dbrg77/scATAC_snakemake.

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Acknowledgements

We thank all members from the Chen lab and Q. Wu for their helpful comments on the manuscript. We thank W. Chen for providing ideas during the revision. We thank C. Hou for sharing reagents. We thank the Flow Cytometry facility in the Department of Biology of Southern University of Science and Technology for the support of single-nucleus/cell sorting. This study was supported by National Key R&D Program of China (2018YFC1004500), National Natural Science Foundation of China (81872330) and Shenzhen Science And Technology Innovation Committee (ZDSYS20200811144002008). The computational work was supported by Center for Computational Science and Engineering at Southern University of Science and Technology.

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Contributions

X.C. conceived the project. W.X. and X.C. designed the protocol. W.X. performed the experiment with the help from Y.L., Q.X. and X.W.. Y.W. and X.C. designed the data processing pipeline and wrote the code. Y.W., W.X. and X.C. analyzed the data. X.C. and W.J. supervised the project. All authors contributed to the writing of the manuscript.

Corresponding author

Correspondence to Xi Chen.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Hiroshi Kimura and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Chen, X. et al. Nat Commun. 9, 5345 (2018): https://doi.org/10.1038/s41467-018-07771-0

Jia, G. et al. Nat. Commun. 9, 4877 (2018): https://doi.org/10.1038/s41467-018-07307-6

Soon, M. et al. Nat. Immunol. 21, 1597–1610 (2020): https://doi.org/10.1038/s41590-020-0800-8

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Xu, W., Wen, Y., Liang, Y. et al. A plate-based single-cell ATAC-seq workflow for fast and robust profiling of chromatin accessibility. Nat Protoc 16, 4084–4107 (2021). https://doi.org/10.1038/s41596-021-00583-5

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