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Cell-type-specific brain methylomes profiled via ultralow-input microfluidics

Abstract

Methylomic analyses typically require substantial amounts of DNA, thus hindering studies involving scarce samples. Here, we show that microfluidic diffusion-based reduced representation bisulfite sequencing (MID-RRBS) permits high-quality methylomic profiling with nanogram-to-single-cell quantities of starting DNA. We used the microfluidic device, which allows for efficient bisulfite conversion with high DNA recovery, to analyse genome-wide DNA methylation in cell nuclei isolated from mouse brains and sorted into NeuN+ (primarily neuronal) and NeuN (primarily glial) fractions, and to establish cell-type-specific methylomes. Genome-wide methylation and methylation in low-CpG-density promoter regions showed distinct patterns for NeuN+ and NeuN fractions from the mouse cerebellum. The identification of substantial variations in the methylomic landscapes of the NeuN+ fraction of the frontal cortex of mice chronically treated with an atypical antipsychotic drug suggests that this technology can be broadly used for cell-type-specific drug profiling and for the study of drug–methylome interactions.

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Fig. 1: MID-RRBS device and protocol.
Fig. 2: MID-RRBS generated high-quality data using less than 1 ng DNA.
Fig. 3: Distinct methylomic features in NeuN+ and NeuN fractions from mouse cerebellum.
Fig. 4: CG methylation levels across various annotated genomic features for NeuN+ and NeuN fractions from mouse cerebellum.
Fig. 5: Pearson’s correlations in CG methylation levels for low-, intermediate- and high-CpG-density promoters (LCP, ICP and HCP) among homogenate, NeuN+ and NeuN samples.
Fig. 6: Interactions between methylomes and transcriptomes for NeuN+ and NeuN from mouse cerebellum.
Fig. 7: Methylomic changes in NeuN+ fraction from mouse frontal cortex associated with chronic clozapine treatment.

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Acknowledgements

We thank C. Luo, J. Lucero, M.M. Behrens and J.R. Ecker at Salk Institute and J. Ma at Carnegie Mellon University for helpful discussion. This work was supported by US National Institutes of Health grants EB017235 (C.L.), HG009256 (C.L.), MH084894 (J.G.-M.), MH111940 (J.G.-M.) and NS094574 (H.X.).

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Authors and Affiliations

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Contributions

C.L. and S.M. designed the microfluidic device. S.M. and C.L. developed the MID-RRBS protocol. C.L. supervised the research. C.L., S.M., J.G.-M. and H.X. designed the biological experiments. S.M. conducted experiments to generate MID-RRBS and mRNA-seq data on the cell/brain samples and performed data analysis. M.d.l.F.R. performed the clozapine treatment and sample collection. Z.S. helped with the device characterization. C.S. and T.W.M. helped with device fabrication and set-up. C.L. and S.M. wrote the manuscript. All authors proofread the manuscript and provided comments.

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Correspondence to Chang Lu.

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Supplementary information

Supplementary Information

Supplementary figures and tables.

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Supplementary Table 5

List of genes associated with differentially CG-methylated regions (10 ng sample).

Supplementary Table 6

List of genes associated with differentially CG-methylated regions (0.5 ng sample).

Supplementary Table 7

GO term enrichment of genes associated with differentially CG methylated regions (10 ng sample).

Supplementary Table 8

GO term enrichment of genes associated with differentially CG methylated regions (0.5 ng sample).

Supplementary Table 10

List of differentially expressed genes in fractions of nuclei extracted from mouse cerebellum.

Supplementary Table 12

List of genes associated with differentially CG-methylated regions in samples from the frontal cortex of clozapine-treated and control mice.

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Ma, S., de la Fuente Revenga, M., Sun, Z. et al. Cell-type-specific brain methylomes profiled via ultralow-input microfluidics. Nat Biomed Eng 2, 183–194 (2018). https://doi.org/10.1038/s41551-018-0204-3

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