Abstract
DNA methylation represents one of the best characterized epigenetic modifications. In particular, global demethylation is a common feature of epigenetic reprogramming to naïve pluripotency in human and mouse pluripotent stem cells. In parallel to the global changes, several locus-specific changes to the DNA methylation landscape occur and also loss of imprinting has been observed in naïve human pluripotent stem cells. The current gold standard to assess and quantitively map DNA methylation is bisulfite sequencing. Various protocols are available for genome-wide bisulfite sequencing and here I describe an optimized method based on Post Bisulfite Adapter Tagging (PBAT) for low amounts of DNA or cells, with as little as 50 cells as minimum requirement, and with the possibility to process a large number of samples in parallel. I outline the basic bioinformatic steps needed to process raw Illumina sequencing data and then describe the inital steps of the analysis of DNA methylation datasets, including an assessment of imprint control regions.
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References
von Meyenn F, Reik W (2015) Forget the parents: epigenetic reprogramming in human germ cells. Cell 161:1248–1251
Reik W, Kelsey G (2014) Epigenetics: cellular memory erased in human embryos. Nature 511:540–541
Kalkan T, Smith A (2014) Mapping the route from naive pluripotency to lineage specification. Philos Trans R Soc Lond B Biol Sci 369:20130540
Takashima Y, Guo G, Loos R et al (2014) Resetting transcription factor control circuitry toward ground-state pluripotency in human. Cell 158:1254–1269
Guo G, von Meyenn F, Santos F et al (2016) Naive pluripotent stem cells derived directly from isolated cells of the human inner cell mass. Stem Cell Rep 6:437–446
Theunissen TW, Powell BE, Wang H et al (2014) Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell 15:471–487
Gafni O, Weinberger L, Mansour AA et al (2013) Derivation of novel human ground state naive pluripotent stem cells. Nature 504:282–286
Guo G, von Meyenn F, Rostovskaya M et al (2017) Epigenetic resetting of human pluripotency. Development 144:2748–2763
Pastor WA, Chen D, Liu W et al (2016) Naive human pluripotent cells feature a methylation landscape devoid of blastocyst or germline memory. Cell Stem Cell 18:323–329
Rostovskaya M, Stirparo GG, Smith A (2019) Capacitation of human naïve pluripotent stem cells for multi-lineage differentiation. Development 146:dev172916
Miura F, Enomoto Y, Dairiki R et al (2012) Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging. Nucleic Acids Res 40:e136
Clark SJ, Smallwood SA, Lee HJ et al (2017) Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq). Nat Protoc 12:534–547
Smallwood SA, Lee HJ, Angermueller C et al (2014) Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat Methods 11:817–820
Frommer M, McDonald LE, Millar DS et al (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A 89:1827–1831
Clark SJ, Harrison J, Paul CL et al (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res 22:2990–2997
Acknowledgement
This protocol is based on method developments and optimizations at the Babraham Institute in Cambridge, UK, and, in particular, on protocols established by Sebastien Smallwood and Stephen Clark.
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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
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von Meyenn, F. (2022). Profiling DNA Methylation in Human Naïve Pluripotent Stem Cells. In: Rugg-Gunn, P. (eds) Human Naïve Pluripotent Stem Cells. Methods in Molecular Biology, vol 2416. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1908-7_11
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DOI: https://doi.org/10.1007/978-1-0716-1908-7_11
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