SpringerLink
Log in

Single-Cell 5fC Sequencing

  • Protocol
  • First Online:
Single Cell Methods

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1979))

Abstract

Active DNA demethylation plays important roles in the epigenetic reprogramming of developmental processes. 5-formylcytosine (5fC) is produced during active demethylation of 5-methylcytosine (5mC). Here, we describe a technique called CLEVER-seq (Chemical-labeling-enabled C-to-T conversion sequencing), which detects the whole genome 5fC distribution at single-base and single-cell resolution. CLEVER-seq is suitable for the analysis of precious samples such as early embryos and laser microdissection captured samples.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
GBP 34.95
Price includes VAT (United Kingdom)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
GBP 87.50
Price includes VAT (United Kingdom)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
GBP 109.99
Price includes VAT (United Kingdom)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free ship** worldwide - see info
Hardcover Book
GBP 179.99
Price includes VAT (United Kingdom)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free ship** worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Kriaucionis S, Heintz N (2009) The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324(5929):929–930. https://doi.org/10.1126/science.1169786. pii: 1169786

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, Agarwal S, Iyer LM, Liu DR, Aravind L, Rao A (2009) Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324(5929):930–935. https://doi.org/10.1126/science.1170116; pii: 1170116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. He YF, Li BZ, Li Z, Liu P, Wang Y, Tang Q, Ding J, Jia Y, Chen Z, Li L, Sun Y, Li X, Dai Q, Song CX, Zhang K, He C, Xu GL (2011) Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333(6047):1303–1307. https://doi.org/10.1126/science.1210944. pii: science.1210944

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ito S, Shen L, Dai Q, Wu SC, Collins LB, Swenberg JA, He C, Zhang Y (2011) Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333(6047):1300–1303. https://doi.org/10.1126/science.1210597; pii: science.1210597

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Maiti A, Drohat AC (2011) Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: potential implications for active demethylation of CpG sites. J Biol Chem 286(41):35334–35338. https://doi.org/10.1074/jbc.C111.284620; pii: C111.284620

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Pfaffeneder T, Hackner B, Truss M, Munzel M, Muller M, Deiml CA, Hagemeier C, Carell T (2011) The discovery of 5-formylcytosine in embryonic stem cell DNA. Angew Chem Int Ed Engl 50(31):7008–7012. https://doi.org/10.1002/anie.201103899

    Article  CAS  PubMed  Google Scholar 

  7. Kohli RM, Zhang Y (2013) TET enzymes, TDG and the dynamics of DNA demethylation. Nature 502(7472):472–479. https://doi.org/10.1038/nature12750

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Pastor WA, Aravind L, Rao A (2013) TETonic shift: biological roles of TET proteins in DNA demethylation and transcription. Nat Rev Mol Cell Biol 14(6):341–356. https://doi.org/10.1038/nrm3589

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wu X, Zhang Y (2017) TET-mediated active DNA demethylation: mechanism, function and beyond. Nat Rev Genet 18(9):517–534. https://doi.org/10.1038/nrg.2017.33

    Article  CAS  PubMed  Google Scholar 

  10. Song CX, Yi C, He C (2012) Map** recently identified nucleotide variants in the genome and transcriptome. Nat Biotechnol 30(11):1107–1116. https://doi.org/10.1038/nbt.2398

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wu H, Zhang Y (2015) Charting oxidized methylcytosines at base resolution. Nat Struct Mol Biol 22(9):656–661. https://doi.org/10.1038/nsmb.3071

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Song CX, Szulwach KE, Dai Q, Fu Y, Mao SQ, Lin L, Street C, Li Y, Poidevin M, Wu H, Gao J, Liu P, Li L, Xu GL, ** P, He C (2013) Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming. Cell 153(3):678–691. https://doi.org/10.1016/j.cell.2013.04.001; pii: S0092-8674(13)00400-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Lu X, Han D, Zhao BS, Song CX, Zhang LS, Dore LC, He C (2015) Base-resolution maps of 5-formylcytosine and 5-carboxylcytosine reveal genome-wide DNA demethylation dynamics. Cell Res 25(3):386–389. https://doi.org/10.1038/cr.2015.5; pii: cr20155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Booth MJ, Marsico G, Bachman M, Beraldi D, Balasubramanian S (2014) Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution. Nat Chem 6(5):435–440. https://doi.org/10.1038/nchem.1893; pii: nchem.1893

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Guo F, Li X, Liang D, Li T, Zhu P, Guo H, Wu X, Wen L, Gu TP, Hu B, Walsh CP, Li J, Tang F, Xu GL (2014) Active and passive demethylation of male and female pronuclear DNA in the mammalian zygote. Cell Stem Cell 15(4):447–458. https://doi.org/10.1016/j.stem.2014.08.003; pii: S1934-5909(14)00341-5

    Article  CAS  PubMed  Google Scholar 

  16. Wu H, Wu X, Shen L, Zhang Y (2014) Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing. Nat Biotechnol 32(12):1231–1240. https://doi.org/10.1038/nbt.3073; pii: nbt.3073

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Neri F, Incarnato D, Krepelova A, Rapelli S, Anselmi F, Parlato C, Medana C, Dal Bello F, Oliviero S (2015) Single-base resolution analysis of 5-formyl and 5-carboxyl cytosine reveals promoter DNA methylation dynamics. Cell Rep. https://doi.org/10.1016/j.celrep.2015.01.008. pii: S2211-1247(15)00009-1

  18. Hu X, Zhang L, Mao SQ, Li Z, Chen J, Zhang RR, Wu HP, Gao J, Guo F, Liu W, Xu GF, Dai HQ, Shi YG, Li X, Hu B, Tang F, Pei D, Xu GL (2014) Tet and TDG mediate DNA demethylation essential for mesenchymal-to-epithelial transition in somatic cell reprogramming. Cell Stem Cell 14(4):512–522. https://doi.org/10.1016/j.stem.2014.01.001

    Article  CAS  PubMed  Google Scholar 

  19. Shen L, Wu H, Diep D, Yamaguchi S, D’Alessio AC, Fung HL, Zhang K, Zhang Y (2013) Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics. Cell 153(3):692–706. https://doi.org/10.1016/j.cell.2013.04.002; pii: S0092-8674(13)00401-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Raiber EA, Beraldi D, Ficz G, Burgess HE, Branco MR, Murat P, Oxley D, Booth MJ, Reik W, Balasubramanian S (2012) Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase. Genome Biol 13(8):R69. https://doi.org/10.1186/gb-2012-13-8-r69; pii: gb-2012-13-8-r69

    Article  PubMed  PubMed Central  Google Scholar 

  21. Iurlaro M, McInroy GR, Burgess HE, Dean W, Raiber EA, Bachman M, Beraldi D, Balasubramanian S, Reik W (2016) In vivo genome-wide profiling reveals a tissue-specific role for 5-formylcytosine. Genome Biol 17(1):141. https://doi.org/10.1186/s13059-016-1001-5

    Article  PubMed  PubMed Central  Google Scholar 

  22. Sun Z, Dai N, Borgaro JG, Quimby A, Sun D, Correa IR Jr, Zheng Y, Zhu Z, Guan S (2015) A sensitive approach to map genome-wide 5-hydroxymethylcytosine and 5-formylcytosine at single-base resolution. Mol Cell 57(4):750–761. https://doi.org/10.1016/j.molcel.2014.12.035; pii: S1097-2765(14)01013-2

    Article  CAS  PubMed  Google Scholar 

  23. **a B, Han D, Lu X, Sun Z, Zhou A, Yin Q, Zeng H, Liu M, Jiang X, **e W, He C, Yi C (2015) Bisulfite-free, base-resolution analysis of 5-formylcytosine at the genome scale. Nat Methods 12(11):1047–1050. https://doi.org/10.1038/nmeth.3569; pii: nmeth.3569

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Wu X, Inoue A, Suzuki T, Zhang Y (2017) Simultaneous map** of active DNA demethylation and sister chromatid exchange in single cells. Genes Dev 31(5):511–523. https://doi.org/10.1101/gad.294843.116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Zhu C, Gao Y, Guo H, **a B, Song J, Wu X, Zeng H, Kee K, Tang F, Yi C (2017) Single-cell 5-formylcytosine landscapes of mammalian early embryos and ESCs at single-base resolution. Cell Stem Cell 20(5):720–731.e725. https://doi.org/10.1016/j.stem.2017.02.013

    Article  CAS  PubMed  Google Scholar 

  26. Zong C, Lu S, Chapman AR, **e XS (2012) Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science 338(6114):1622–1626. https://doi.org/10.1126/science.1229164; pii: 338/6114/1622

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

The authors thank Dr. Hongshan Guo, Bo ** the original protocol. Part of the analysis was performed on the Computing Platform of the Center for Life Sciences. This work was supported by the National Basic Research Program of China and the National Natural Science Foundation of China (91519325, MOST2016YFC0900300, 21522201, and 2014CB964900).

Competing financial interests: C.Z. and C.Y. are coinventors on filed patents (201710111600.9) for the labeling strategy and sequencing method reported herein.

Author information

Authors and Affiliations

  1. State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Bei**g, People’s Republic of China

    Chenxu Zhu, **ying Peng & Chengqi Yi

  2. Biodynamic Optical Imaging Center, Bei**g Advanced Innovation Center for Genomics, School of Life Sciences, Peking University, Bei**g, People’s Republic of China

    Yun Gao & Fuchou Tang

  3. Peking-Tsinghua Center for Life Sciences, Peking University, Bei**g, People’s Republic of China

    Fuchou Tang & Chengqi Yi

  4. Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Peking University, Bei**g, People’s Republic of China

    Fuchou Tang

  5. Department of Chemical Biology and Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Bei**g, People’s Republic of China

    Chengqi Yi

Authors
  1. Chenxu Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yun Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. **ying Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fuchou Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chengqi Yi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Chengqi Yi .

Editor information

Editors and Affiliations

  1. Department of Life Sciences and System Biology, University of Turin, Italian Institute for Genomic Medicine, IIGM Turin, Torino, Italy

    Valentina Proserpio

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Zhu, C., Gao, Y., Peng, J., Tang, F., Yi, C. (2019). Single-Cell 5fC Sequencing. In: Proserpio, V. (eds) Single Cell Methods. Methods in Molecular Biology, vol 1979. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9240-9_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9240-9_16

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9239-3

  • Online ISBN: 978-1-4939-9240-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation