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Chromatin accessibility profiling by ATAC-seq

Nature Protocols volume 17, pages 1518–1552 (2022)Cite this article

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Abstract

The assay for transposase-accessible chromatin using sequencing (ATAC-seq) provides a simple and scalable way to detect the unique chromatin landscape associated with a cell type and how it may be altered by perturbation or disease. ATAC-seq requires a relatively small number of input cells and does not require a priori knowledge of the epigenetic marks or transcription factors governing the dynamics of the system. Here we describe an updated and optimized protocol for ATAC-seq, called Omni-ATAC, that is applicable across a broad range of cell and tissue types. The ATAC-seq workflow has five main steps: sample preparation, transposition, library preparation, sequencing and data analysis. This protocol details the steps to generate and sequence ATAC-seq libraries, with recommendations for sample preparation and downstream bioinformatic analysis. ATAC-seq libraries for roughly 12 samples can be generated in 10 h by someone familiar with basic molecular biology, and downstream sequencing analysis can be implemented using benchmarked pipelines by someone with basic bioinformatics skills and with access to a high-performance computing environment.

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Fig. 1: Schematic of the ATAC-seq transposition reaction and library preparation.
Fig. 2: Schematic overview of ATAC-seq protocol.
Fig. 3: Assessing ATAC-seq library quality.
Fig. 4: Overview of the steps of ATAC-seq data analysis.
Fig. 5: Schematic of peak merging strategies and the resulting merged peak sets.

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Data availability

The ATAC-seq datasets generated for the protocol optimizations detailed in Supplementary Figs. 1, 4 and 5 are available on the Gene Expression Omnibus under accession number GSE188797. The data used in Fig. 5 are taken from ref. 38. All analyses were performed using the hg38 human genome.

Code availability

The source code for the iterative overlap is freely available at https://github.com/corceslab/ATAC_IterativeOverlapPeakMerging88. All other ATAC-seq data analysis for the figures used in this protocol were generated using PEPATAC73 with Bulker (container version 1.0.8), available at http://pepatac.databio.org/en/latest/.

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Acknowledgements

This work was supported by NIH R00-AG059918, U01-AG072573, P01-AG073082, UM1-HG012076 and a gift from the Ray and Dagmar Dolby Family Fund (to the Gladstone Institutes). F.C.G. is an Alan Kaganov Scholar. M.R.C. is additionally supported by the Farmer Family Foundation Parkinson’s Research Initiative and an American Society of Hematology Scholar Award.

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Authors and Affiliations

  1. Gladstone Institute of Neurological Disease, San Francisco, CA, USA

    Fiorella C. Grandi, Hailey Modi, Lucas Kampman & M. Ryan Corces

  2. Gladstone Institute of Data Science and Biotechnology, San Francisco, CA, USA

    Fiorella C. Grandi, Hailey Modi, Lucas Kampman & M. Ryan Corces

  3. Department of Neurology, University of California San Francisco, San Francisco, CA, USA

    Fiorella C. Grandi, Hailey Modi, Lucas Kampman & M. Ryan Corces

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  1. Fiorella C. Grandi
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  2. Hailey Modi
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  3. Lucas Kampman
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Contributions

All authors contributed to developing this protocol. All experiments were performed by H.M., F.C.G. and L.K. with supervision from M.R.C. The manuscript was written by F.C.G., L.K. and H.M. with input from all authors.

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Correspondence to M. Ryan Corces.

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The authors declare no competing interests.

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Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Corces, M. R. et al. Nat. Methods 14, 959–962 (2017): https://doi.org/10.1038/nmeth.4396

Corces, M. R. et al. Science 362, 6413 (2018): https://doi.org/10.1126/science.aav1898

Corces, M. R. et al. Nat. Genet. 52, 1158–1168 (2020): https://doi.org/10.1038/s41588-020-00721-x

Supplementary information

Supplementary Information

Supplementary Figs. 1–6, Supplementary Notes 1–4, Supplementary Protocol 1 (Nuclei Isolation), Supplementary Methods and Supplementary References.

Supplementary Tables 1 and 2

Alignment statistics for different ATAC-seq library read lengths. ATAC-seq dual-indexing adapters and barcode sequences.

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Grandi, F.C., Modi, H., Kampman, L. et al. Chromatin accessibility profiling by ATAC-seq. Nat Protoc 17, 1518–1552 (2022). https://doi.org/10.1038/s41596-022-00692-9

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