ChIP-Sequencing to Map the Epigenome of Senescent Cells Using Benzonase Endonuclease

T. S. Rai, P. D. Adams

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Citation (Scopus)

Abstract

Cellular senescence is a state of stable cell cycle arrest triggered by diverse stresses. Establishment of senescence occurs in conjunction with a multitude of chromatin changes, which are just beginning to be studied. These chromatin changes are hypothesized to be causative for senescence. Currently, a preferred method to study such changes is chromatin immunoprecipitation followed by sequencing (ChIP-Seq). This is usually done by cross-linking the cells with formaldehyde and then generating chromatin fragments between 150 and 300 bp by sonication. The DNA replication-independent histone chaperone HIRA plays an important role in control of chromatin in nonproliferating senescent cells. While investigating the role of HIRA in senescence, we found conventional ChIP protocols to be problematic, routinely yielding too low amounts of DNA for sequencing. To overcome these problems we adapted and optimized an alternative ChIP method that does not rely on cross-linking and sonication for chromatin fragmentation, and is able to easily isolate chromatin from senescent cells ready for immunoprecipitation. This method uses Benzonase endonuclease for solubilization of uncross-linked chromatin by digestion of DNA and RNA, in the absence of proteolytic activity. Using this protocol, we were easily able to immunoprecipitate HIRA with sufficient DNA for Illumina sequencing.

LanguageEnglish
Title of host publicationEnzymes of Epigenetics, Part B
Pages355-364
Number of pages10
Volume574
DOIs
Publication statusPublished - 1 Jan 2016

Publication series

NameMethods in Enzymology
Volume574
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Fingerprint

Serratia marcescens nuclease
Endonucleases
Chromatin
Sonication
DNA Sequence Analysis
DNA
Histone Chaperones
Chromatin Immunoprecipitation
Cell Aging
Cell Cycle Checkpoints
DNA Replication
Immunoprecipitation
Formaldehyde
Digestion

Keywords

  • Benzonase
  • ChIP
  • Helium
  • HIRA
  • Senescence

Cite this

Rai, T. S., & Adams, P. D. (2016). ChIP-Sequencing to Map the Epigenome of Senescent Cells Using Benzonase Endonuclease. In Enzymes of Epigenetics, Part B (Vol. 574, pp. 355-364). (Methods in Enzymology; Vol. 574). https://doi.org/10.1016/bs.mie.2016.01.021
Rai, T. S. ; Adams, P. D. / ChIP-Sequencing to Map the Epigenome of Senescent Cells Using Benzonase Endonuclease. Enzymes of Epigenetics, Part B. Vol. 574 2016. pp. 355-364 (Methods in Enzymology).
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Rai, TS & Adams, PD 2016, ChIP-Sequencing to Map the Epigenome of Senescent Cells Using Benzonase Endonuclease. in Enzymes of Epigenetics, Part B. vol. 574, Methods in Enzymology, vol. 574, pp. 355-364. https://doi.org/10.1016/bs.mie.2016.01.021

ChIP-Sequencing to Map the Epigenome of Senescent Cells Using Benzonase Endonuclease. / Rai, T. S.; Adams, P. D.

Enzymes of Epigenetics, Part B. Vol. 574 2016. p. 355-364 (Methods in Enzymology; Vol. 574).

Research output: Chapter in Book/Report/Conference proceedingChapter

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Rai TS, Adams PD. ChIP-Sequencing to Map the Epigenome of Senescent Cells Using Benzonase Endonuclease. In Enzymes of Epigenetics, Part B. Vol. 574. 2016. p. 355-364. (Methods in Enzymology). https://doi.org/10.1016/bs.mie.2016.01.021