Genome-wide profiling of nucleosome position and chromatin accessibility in single cells using scMNase-seq

Abstract

Nucleosome organization is important for chromatin compaction and accessibility. Profiling nucleosome positioning genome-wide in single cells provides critical information to understand the cell-to-cell heterogeneity of chromatin states within a cell population. This protocol describes single-cell micrococcal nuclease sequencing (scMNase-seq), a method for detecting genome-wide nucleosome positioning and chromatin accessibility simultaneously from a small number of cells or single cells. To generate scMNase-seq libraries, single cells are isolated by FACS sorting, lysed and digested by MNase. DNA is purified, end-repaired and ligated to Y-shaped adaptors. Following PCR amplification with indexing primers, the subnucleosome-sized (fragments with a length of ≤80 bp) and mononucleosome-sized (fragments with a length between 140 and 180 bp) DNA fragments are recovered and sequenced on Illumina HiSeq platforms. On average, 0.5–1 million unique mapped reads are obtained for each single cell. The mononucleosome-sized DNA fragments precisely define genome-wide nucleosome positions in single cells, while the subnucleosome-sized DNA fragments provide information on chromatin accessibility. Library preparation of scMNase-seq takes only 2 d, requires only standard molecular biology techniques and does not require sophisticated laboratory equipment. Processing of high-throughput sequencing data requires basic bioinformatics skills and uses publicly available bioinformatics software.

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Fig. 1: Workflow illustration of the scMNase-seq protocol.
Fig. 2: Dead or apoptotic cells exhibit chromatin fragmentation without MNase digestion.
Fig. 3: MNase titration using bulk cells.
Fig. 4: Gel image showing the size range of scMNase-seq library DNA after PCR amplification.
Fig. 5: Average profiling of nucleosomes and subnucleosomes from pooled single cells.

Data availability

Data used in this protocol have been deposited in the Gene Expression Omnibus database with accession number GSE96688. Figure 5 is associated with the raw data. There are no restrictions on data availability.

Code availability

Codes used in this protocol have been deposited in GitHub (https://github.com/binbinlai2012/scMNase). There are no restrictions on code availability.

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Acknowledgements

We thank the National Heart, Lung, and Blood Institute DNA Sequencing Core Facility for sequencing the libraries and the National Heart, Lung, and Blood Institute Flow Cytometry Core facility for sorting the cells. The work was supported by the Division of Intramural Research, National Heart, Lung and Blood Institute (K.Z.), the National Key Research and Development Project (no. 2016YFA0502203) (B.N.), the General Program of National Natural Science Foundation of China (no. 81670534) (B.N.), and the Medical Scientific and Technological Innovation Funds of Southwest Hospital (no. SWH2016LHYS-04) (B.N.).

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Authors

Contributions

K.Z. and B.N. directed the project. W.G. performed the experiments. B.L. analyzed the data. W.G., B.L. and K.Z. wrote the manuscript.

Corresponding authors

Correspondence to Bing Ni or Keji Zhao.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Gonçalo Castelo-Branco, Emily Hodges 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.

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Key references using this protocol

Lai, B. et al. Nature 562, 281–285 (2018): https://doi.org/10.1038/s41586-018-0567-3

Integrated supplementary information

Supplementary Fig. 1 FACS sorting of single cells by FSC versus SSC gating.

Formaldehyde-fixed mouse naïve T cells (100,000) were submitted for FACS sorting based on size and granularity (FSC vs SSC, P1 gate), and cell doublets were excluded firstly by SSC-H vs SSC-W (P2 gate) and further by FSC-H vs FSC-W (P3 gate).

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Supplementary Table 1.

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Gao, W., Lai, B., Ni, B. et al. Genome-wide profiling of nucleosome position and chromatin accessibility in single cells using scMNase-seq. Nat Protoc 15, 68–85 (2020). https://doi.org/10.1038/s41596-019-0243-6

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