Using Native Chromatin Immunoprecipitation to Interrogate Histone Variant Protein Deposition in Embryonic Stem Cells

Methods Mol Biol. 2014;1176:11-22. doi: 10.1007/978-1-4939-0992-6_2.

Abstract

Chromatin immunoprecipitation combined with massive parallel sequencing (ChIP-Seq) is a powerful epigenetics technique for interrogating the genome-wide localization of histone modifications, histone variants, and other chromatin-associating factors. In brief, chromatin pellets are fractionated from the nuclei, and then fragmented by enzymatic digestion or sonication. Chromatin regions associated with proteins of interest are enriched by immunoprecipitation with specific antibodies. After the immunoprecipitation, DNA fragments are extracted, amplified during sequencing library construction, and sequenced by high-throughput sequencing. Here, we describe the native chromatin immunoprecipitation of a rare histone variant, H2A.X, followed by high-throughput sequencing, in mouse embryonic stem cells.

MeSH terms

  • Animals
  • Chromatin / genetics
  • Chromatin / metabolism*
  • Chromatin Immunoprecipitation / methods*
  • Embryonic Stem Cells / metabolism*
  • Epigenesis, Genetic
  • Epigenomics / methods
  • Gene Library
  • High-Throughput Nucleotide Sequencing
  • Histones / metabolism*
  • Mice

Substances

  • Chromatin
  • H2AX protein, mouse
  • Histones