Abstract
ChIP-seq, or chromatin immunoprecipitation combined with massively parallel DNA sequencing, is a powerful technique to investigate in vivo protein–DNA interactions on a genome-wide scale at high resolution. Here we describe a ChIP-seq protocol optimized for analysis of condensin I complex on human mitotic chromosomes. The protocol includes procedures of intensive cell fixation by two cross-linking reagents and thorough chromatin shearing by nuclease and sonication treatments, both of which contribute to improving the signal-to-noise ratio of condensin I ChIP-seq profiles. The optimized protocol may also be helpful to explore chromosomal binding sites of other “hard-to-see” proteins by ChIP-seq.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Park PJ (2009) ChIP-seq: advantages and challenges of a maturing technology. Nat Rev Genet 10:669–680
Deardorff MA, Bando M, Nakato R et al (2012) HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle. Nature 489:313–317
Kagey MH, Newman JJ, Bilodeau S et al (2011) Mediator and cohesin connect gene expression and chromatin architecture. Nature 472:247
Schmidt D, Schwalie PC, Ross-Innes CS et al (2010) A CTCF-independent role for cohesin in tissue-specific transcription. Genome Res 20:578–588
Sutani T, Sakata T, Nakato R et al (2015) Condensin targets and reduces unwound DNA structures associated with transcription in mitotic chromosome condensation. Nat Commun 6:7815
Komata M, Katou Y, Tanaka H et al (2014) Chromatin Immunoprecipitation Protocol for Mammalian Cells. In: Miyamoto-Sato E, Ohashi H, Sasaki H et al (eds) Transcription factor regulatory networks. The series of methods in molecular biology, vol 1164. Springer, New York, NY, pp 33–38
Kidder BL, Hu G, Zhao K (2011) ChIP-Seq: technical considerations for obtaining high-quality data. Nat Immunol 12:918–922
Gerlich D, Hirota T, Koch B et al (2006) Condensin I stabilizes chromosomes mechanically through a dynamic interaction in live cells. Curr Biol 16:333–344
Poorey K, Viswanathan R, Carver MN et al (2013) Measuring chromatin interaction dynamics on the second time scale at single-copy genes. Science 342:369–372
Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25
Nakato R, Itoh T, Shirahige K (2013) DROMPA: easy-to-handle peak calling and visualization software for the computational analysis and validation of ChIP-seq data. Genes Cells 18:589–601
Robinson JT, Thorvaldsdóttir H, Winckler W et al (2011) Integrative genomics viewer. Nat Biotechnol 29:24–26
Thorvaldsdottir H, Robinson JT, Mesirov JP (2013) Integrative genomics viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 14:178–192
Kent WJ, Sugnet CW, Furey TS et al (2002) The human genome browser at UCSC. Genome Res 12:996–1006
Kurdistani S (2003) In vivo protein–protein and protein–DNA crosslinking for genomewide binding microarray. Methods 31:90–95
Nowak D, Tian B, Brasier A (2005) Two-step cross-linking method for identification of NF-kB gene network by chromatin immunoprecipitation. Biotechniques 39:715–725
Zhang J, Poh HM, Peh SQ et al (2012) ChIA-PET analysis of transcriptional chromatin interactions. Methods 58:289–299
Landt SG, Marinov GK, Kundaje A et al (2012) ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia. Genome Res 22:1813–1831
Kharchenko PV, Tolstorukov MY, Park PJ (2008) Design and analysis of ChIP-seq experiments for DNA-binding proteins. Nat Biotechnol 26:1351–1359
Pepke S, Wold B, Mortazavi A (2009) Computation for ChIP-seq and RNA-seq studies. Nat Methods 6:S22–S32
Acknowledgment
We thank Y. Katou, R. Nakato, M. Bando, and all other members of the Shirahige laboratory for support and discussion. This work was supported in part by CREST from JST (K.S.), by Grants-in-aid for Scientific Research (A) (K.S.), Grants-in-aid for Scientific Research (C) (T.Su.), and Grants-in-aid for Scientific Research on Innovative Areas (K.S.) from JSPS (KAKENHI grant numbers 15H02369, 24570006, and 15H05976, respectively). T.Sa. is supported by JSPS research fellowship for young scientists.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Sakata, T., Shirahige, K., Sutani, T. (2017). ChIP-seq Analysis of Condensin Complex in Cultured Mammalian Cells. In: Yokomori, K., Shirahige, K. (eds) Cohesin and Condensin. Methods in Molecular Biology, vol 1515. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6545-8_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6545-8_16
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6543-4
Online ISBN: 978-1-4939-6545-8
eBook Packages: Springer Protocols