title: ChIP-Sequencing to Map the Epigenome of Senescent Cells Using Benzonase Endonuclease.
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 300bp 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.
authors.affiliation: Institute of Biomedical and Environmental Health Research, University of the West of Scotland, Paisley, United Kingdom. Electronic address: taranjitsingh.rai@uws.ac.uk.
authors.forename: T S
authors.initials: TS
authors.lastname: Rai
authors.affiliation: CR-UK Beatson Labs, Institute of Cancer Sciences, University of Glasgow, Glasgow, United Kingdom. Electronic address: p.adams@beatson.gla.ac.uk.
authors.forename: P D
authors.initials: PD
authors.lastname: Adams
mesh_terms: D000818:Animals; D059447:Cell Cycle Checkpoints; D016922:Cellular Senescence; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004722:Endoribonucleases / Q000378:metabolism*; D057890:Epigenomics / Q000379:methods*; D006801:Humans; D017422:Sequence Analysis, DNA / Q000379:methods; D012706:Serratia marcescens / Q000201:enzymology*
chemical_list: D002843:Chromatin; D004247:DNA; D004706:Endodeoxyribonucleases; D004722:Endoribonucleases; C021835:Serratia marcescens nuclease
keywords: Benzonase; ChIP; HIRA; Helium; Senescence