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Genome-wide mapping of DNase I hypersensitive sites in rare cell populations using single-cell DNase sequencing

Nature Protocols volume 12, pages 2342–2354 (2017)Cite this article

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Abstract

Increased chromatin accessibility is a feature of cell-type-specific cis-regulatory elements; therefore, mapping of DNase I hypersensitive sites (DHSs) enables the detection of active regulatory elements of transcription, including promoters, enhancers, insulators and locus-control regions. Single-cell DNase sequencing (scDNase-seq) is a method of detecting genome-wide DHSs when starting with either single cells or <1,000 cells from primary cell sources. This technique enables genome-wide mapping of hypersensitive sites in a wide range of cell populations that cannot be analyzed using conventional DNase I sequencing because of the requirement for millions of starting cells. Fresh cells, formaldehyde-cross-linked cells or cells recovered from formalin-fixed paraffin-embedded (FFPE) tissue slides are suitable for scDNase-seq assays. To generate scDNase-seq libraries, cells are lysed and then digested with DNase I. Circular carrier plasmid DNA is included during subsequent DNA purification and library preparation steps to prevent loss of the small quantity of DHS DNA. Libraries are generated for high-throughput sequencing on the Illumina platform using standard methods. Preparation of scDNase-seq libraries requires only 2 d. The materials and molecular biology techniques described in this protocol should be accessible to any general molecular biology laboratory. Processing of high-throughput sequencing data requires basic bioinformatics skills and uses publicly available bioinformatics software.

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Figure 1: Results of pilot experiment to determine optimal DNase I enzyme concentration.
Figure 2: Use of proximal and distal DHS read enrichment to determine optimal DNase I enzyme concentration.
Figure 3: Gel image demonstrating the importance of excluding dead or apoptotic cells before DNase I digestion.
Figure 4: Representative IGV genome browser images of scDNase-seq experiments using cells of differing viabilities22.
Figure 5: Use of shorter fragments improves enrichment at the TSS.
Figure 6: scDNase-seq library quality can be estimated by TSS enrichment profiles.

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Acknowledgements

We thank W. Jin for his insights into the bioinformatics analysis of the data, the National Heart, Lung, and Blood Institute (NHLBI) DNA Sequencing Core facility for sequencing the libraries and the NHLBI Flow Cytometry Core facility for sorting the cells. The work was supported by the Division of Intramural Research, NHLBI (K.Z.).

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Authors and Affiliations

  1. Division of Intramural Research, Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA

    James Cooper, Yi Ding & Keji Zhao

  2. Division of Intramural Research, Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA

    James Cooper

  3. Department of Animal and Avian Sciences, University of Maryland, College Park, Maryland, USA

    Yi Ding & Jiuzhou Song

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  1. James Cooper
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Contributions

J.C. and K.Z. wrote the manuscript. J.C., Y.D., J.S. and K.Z planned and designed the experiments. J.C., Y.D. and K.Z. performed the experiments.

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Correspondence to Keji Zhao.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Library adaptor and multiplex PCR primer sequences. The table lists the DNA sequences for the library adaptors and PCR multiplexing primers compatible with the Illumina sequencing platform. The sequence information is derived from the Illumina Adapter Sequences Document (1000000002694 v01). When ordering the PCR primers, the final 3′ thymine should have a modified phosphorothioate bond (denoted by “*” in the table) to reduce exonuclease degradation. In addition, the 5′ end of the Illumina multiplexing adaptor-top should be phosphorylated (denoted by “P-” in the table). (XLSX 10 kb)

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Cooper, J., Ding, Y., Song, J. et al. Genome-wide mapping of DNase I hypersensitive sites in rare cell populations using single-cell DNase sequencing. Nat Protoc 12, 2342–2354 (2017). https://doi.org/10.1038/nprot.2017.099

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