Genome-wide mapping of endogenous G-quadruplex DNA structures by chromatin immunoprecipitation and high-throughput sequencing


G-rich DNA sequences can form four-stranded G-quadruplex (G4) secondary structures and are linked to fundamental biological processes such as transcription, replication and telomere maintenance. G4s are also implicated in promoting genome instability, cancer and other diseases. Here, we describe a detailed G4 ChIP-seq method that robustly enables the determination of G4 structure formation genome-wide in chromatin. This protocol adapts traditional ChIP-seq for the detection of DNA secondary structures through the use of a G4-structure-specific single-chain antibody with refinements in chromatin immunoprecipitation followed by high-throughput sequencing. This technology does not require expression of the G4 antibody in situ, enabling broad applicability to theoretically all chromatin sources. Beginning with chromatin isolation and antibody preparation, the entire protocol can be completed in <1 week, including basic computational analysis.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: General G4 ChIP-seq workflow.
Figure 3: BG4 quality assessment.
Figure 2: Expected results from a typical G4 ChIP-seq experiment.

Accession codes

Primary accessions

Gene Expression Omnibus


  1. 1

    Thurman, R.E. et al. The accessible chromatin landscape of the human genome. Nature 489, 75–82 (2012).

    CAS  Article  Google Scholar 

  2. 2

    Bochman, M.L., Paeschke, K. & Zakian, V.A. DNA secondary structures: stability and function of G-quadruplex structures. Nat. Rev. Genet. 13, 770–80 (2012).

    CAS  Article  Google Scholar 

  3. 3

    Huppert, J.L. & Balasubramanian, S. Prevalence of quadruplexes in the human genome. Nucleic Acids Res. 33, 2908–2916 (2005).

    CAS  Article  Google Scholar 

  4. 4

    Bedrat, A., Lacroix, L. & Mergny, J.L. Re-evaluation of G-quadruplex propensity with G4Hunter. Nucleic Acids Res. 44, 1746–1759 (2016).

    Article  Google Scholar 

  5. 5

    Paeschke, K. et al. Telomerase recruitment by the telomere end binding protein-beta facilitates G-quadruplex DNA unfolding in ciliates. Nat. Struct. Mol. Biol. 15, 598–604 (2008).

    CAS  Article  Google Scholar 

  6. 6

    Ribeyre, C. et al. The yeast Pif1 helicase prevents genomic instability caused by G-quadruplex-forming CEB1 sequences in vivo. PLoS Genet. 5 e1000475 (2009).

    Article  Google Scholar 

  7. 7

    Paeschke, K. et al. Pif1 family helicases suppress genome instability at G-quadruplex motifs. Nature 497, 458–462 (2013).

    CAS  Article  Google Scholar 

  8. 8

    Vannier, J.B., Pavicic-Kaltenbrunner, V., Petalcorin, M.I.R., Ding, H. & Boulton, S.J. RTEL1 dismantles T loops and counteracts telomeric G4-DNA to maintain telomere integrity. Cell 149, 795–806 (2012).

    CAS  Article  Google Scholar 

  9. 9

    Sarkies, P., Reams, C., Simpson, L.J. & Sale, J.E. Epigenetic instability due to defective replication of structured DNA. Mol. Cell 40, 703–713 (2010).

    CAS  Article  Google Scholar 

  10. 10

    Siddiqui-Jain, A., Grand, C.L., Bearss, D.J. & Hurley, L.H. Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription. Proc. Natl. Acad. Sci. USA 99, 11593–11598 (2002).

    CAS  Article  Google Scholar 

  11. 11

    Rodriguez, R. et al. Small-molecule-induced DNA damage identifies alternative DNA structures in human genes. Nat. Chem. Biol. 8, 301–310 (2012).

    CAS  Article  Google Scholar 

  12. 12

    Mendoza, O., Bourdoncle, A., Boulé, J.B., Brosh, R.M. & Mergny, J.L. G-quadruplexes and helicases. Nucleic Acids Res. 44, 1989–2006 (2016).

    CAS  Article  Google Scholar 

  13. 13

    Maizels, N. & Gray, L.T. The G4 genome. PLoS Genet. 9, e1003468 (2013).

    CAS  Article  Google Scholar 

  14. 14

    Rhodes, D. & Lipps, H.J. G-quadruplexes and their regulatory roles in biology. Nucleic Acids Res. 43, 8627–8637 (2015).

    CAS  Article  Google Scholar 

  15. 15

    Hänsel-Hertsch, R., Di Antonio, M. & Balasubramanian, S. DNA G-quadruplexes in the human genome: detection, functions and therapeutic potential. Nat. Rev. Mol. Cell Biol. 18, 279–284 (2017).

    Article  Google Scholar 

  16. 16

    Schaffitzel, C. et al. In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei. Proc. Natl. Acad. Sci. USA 98, 8572–8577 (2001).

    CAS  Article  Google Scholar 

  17. 17

    Biffi, G., Tannahill, D., McCafferty, J. & Balasubramanian, S. Quantitative visualization of DNA G-quadruplex structures in human cells. Nat. Chem. 5, 182–186 (2013).

    CAS  Article  Google Scholar 

  18. 18

    Biffi, G., Tannahill, D., Miller, J., Howat, W.J. & Balasubramanian, S. Elevated levels of G-quadruplex formation in human stomach and liver cancer tissues. PLoS One 9, e102711 (2014).

    Article  Google Scholar 

  19. 19

    Henderson, A. et al. Detection of G-quadruplex DNA in mammalian cells. Nucleic Acids Res. 42, 860–869 (2014).

    CAS  Article  Google Scholar 

  20. 20

    Gray, L.T., Vallur, A.C., Eddy, J. & Maizels, N. G quadruplexes are genomewide targets of transcriptional helicases XPB and XPD. Nat. Chem. Biol. 10, 313–318 (2014).

    CAS  Article  Google Scholar 

  21. 21

    Neidle, S. Quadruplex nucleic acids as novel therapeutic targets. J. Med. Chem. 59, 5987–6011 (2016).

    CAS  Article  Google Scholar 

  22. 22

    Chambers, V.S. et al. High-throughput sequencing of DNA G-quadruplex structures in the human genome. Nat. Biotechnol. 33, 877–881 (2015).

    Article  Google Scholar 

  23. 23

    Mukundan, V.T. Bulges in G4 quadruplexes: broadening the definition of G4 quadruplex-forming sequences. J. Am. Chem. Soc. 135, 5017–5028 (2013).

    CAS  Article  Google Scholar 

  24. 24

    Hänsel-Hertsch, R. et al. G-quadruplex structures mark human regulatory chromatin. Nat. Genet. 48, 1267–1272 (2016).

    Article  Google Scholar 

  25. 25

    Lam, E.Y.N., Beraldi, D., Tannahill, D. & Balasubramanian, S. G-quadruplex structures are stable and detectable in human genomic DNA. Nat. Commun. 4, 1796 (2013).

    Article  Google Scholar 

  26. 26

    Xu, H. et al. CX-5461 is a DNA G-quadruplex stabilizer with selective lethality in BRCA1/2 deficient tumours. Nat. Commun. 8, 14432 (2017).

    CAS  Article  Google Scholar 

  27. 27

    Law, M.J. et al. ATR-X syndrome protein targets tandem repeats and influences allele-specific expression in a size-dependent manner. Cell 143, 367–378 (2010).

    CAS  Article  Google Scholar 

  28. 28

    Kanoh, Y. et al. Rif1 binds to G quadruplexes and suppresses replication over long distances. Nat. Struct. Mol. Biol. 22, 889–897 (2015).

    CAS  Article  Google Scholar 

  29. 29

    Paeschke, K., Capra, J.A. & Zakian, V.A. DNA replication through G-quadruplex motifs is promoted by the Saccharomyces cerevisiae Pif1 DNA helicase. Cell 145, 678–691 (2011).

    CAS  Article  Google Scholar 

  30. 30

    Liu, H. et al. Conformation selective antibody enables genome profiling and leads to discovery of parallel G-quadruplex in human telomeres. Cell Chem. Biol. 23, 1261–1270 (2016).

    CAS  Article  Google Scholar 

  31. 31

    Furey, T.S. ChIP-seq and beyond: new and improved methodologies to detect and characterize protein-DNA interactions. Nat. Rev. Genet. 13, 840–852 (2012).

    CAS  Article  Google Scholar 

  32. 32

    Ginno, P.A., Lott, P.L., Christensen, H.C., Korf, I. & Chédin, F. R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters. Mol. Cell 45, 814–825 (2012).

    CAS  Article  Google Scholar 

  33. 33

    Adey, A. et al. Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition. Genome Biol. 11, R119 (2010).

    CAS  Article  Google Scholar 

  34. 34

    Teytelman, L. et al. Impact of chromatin structures on DNA processing for genomic analyses. PLoS One 4, e6700 (2009).

    Article  Google Scholar 

  35. 35

    Meyer, C.A. & Liu, X.S. Identifying and mitigating bias in next-generation sequencing methods for chromatin biology. Nat. Rev. Genet. 15, 709–721 (2014).

    CAS  Article  Google Scholar 

  36. 36

    Rhee, H.S. & Pugh, B.F. Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution. Cell 147, 1408–1419 (2011).

    CAS  Article  Google Scholar 

  37. 37

    Rotem, A. et al. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state. Nat. Biotechnol. 33, 1165–1172 (2015).

    CAS  Article  Google Scholar 

  38. 38

    Schmidl, C., Rendeiro, A.F., Sheffield, N.C. & Bock, C. ChIPmentation: fast, robust, low-input ChIP-seq for histones and transcription factors. Nat. Methods 12, 963–5 (2015).

    CAS  Article  Google Scholar 

  39. 39

    Machanick, P. & Bailey, T.L. MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics 27, 1696–1697 (2011).

    CAS  Article  Google Scholar 

  40. 40

    Grant, C.E., Bailey, T.L. & Noble, W.S. FIMO: scanning for occurrences of a given motif. Bioinformatics 27, 1017–1018 (2011).

    CAS  Article  Google Scholar 

  41. 41

    Schmidt, D. et al. ChIP-seq: using high-throughput sequencing to discover protein-DNA interactions. Methods 48, 240–248 (2009).

    CAS  Article  Google Scholar 

  42. 42

    Nakato, R. & Shirahige, K. Recent advances in ChIP-seq analysis: from quality management to whole-genome annotation. Brief Bioinform. 18, 279–290 (2016).

    PubMed Central  Google Scholar 

  43. 43

    Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17, 10 (2011).

    Article  Google Scholar 

  44. 44

    Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Preprint at arXiv, (2013).

  45. 45

    Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  46. 46

    Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    Article  Google Scholar 

  47. 47

    Kent, W.J. et al. The Human Genome Browser at UCSC. Genome Res. 12, 996–1006 (2002).

    CAS  Article  Google Scholar 

  48. 48

    Robinson, J.T. et al. Integrative Genome Viewer. Nat. Biotechnol. 29, 24–6 (2011).

    CAS  Article  Google Scholar 

  49. 49

    Ramírez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44, W160–W165 (2016).

    Article  Google Scholar 

  50. 50

    Wanrooij, P.H. et al. A hybrid G-quadruplex structure formed between RNA and DNA explains the extraordinary stability of the mitochondrial R-loop. Nucleic Acids Res. 40, 10334–10344 (2012).

    CAS  Article  Google Scholar 

  51. 51

    Arrigoni, L. et al. Standardizing chromatin research: a simple and universal method for ChIP-seq. Nucleic Acids Res. 44, e67 (2016).

    Article  Google Scholar 

Download references


We acknowledge support from the University of Cambridge and Cancer Research UK. The Balasubramanian laboratory is supported by core funding from Cancer Research UK (C14303/A17197). S.B. is a Senior Investigator of the Wellcome Trust (grant no. 099232/z/12/z). J.S. is a Marie Curie Fellow of the European Union (747297-QAPs-H2020-MSCA-IF-2016).

Author information




R.H.-H. developed the G4 ChIP-seq method. R.H.-H. and J.S. performed the experiments. R.H.-H., J.S. and G.M. performed bioinformatics analysis. All authors interpreted the data and wrote the manuscript.

Corresponding author

Correspondence to Shankar Balasubramanian.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hänsel-Hertsch, R., Spiegel, J., Marsico, G. et al. Genome-wide mapping of endogenous G-quadruplex DNA structures by chromatin immunoprecipitation and high-throughput sequencing. Nat Protoc 13, 551–564 (2018).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.