Standardizing chromatin research: a simple and universal method for ChIP-seq

Nucleic Acids Res. 2016 Apr 20;44(7):e67. doi: 10.1093/nar/gkv1495. Epub 2015 Dec 23.

Abstract

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a key technique in chromatin research. Although heavily applied, existing ChIP-seq protocols are often highly fine-tuned workflows, optimized for specific experimental requirements. Especially the initial steps of ChIP-seq, particularly chromatin shearing, are deemed to be exceedingly cell-type-specific, thus impeding any protocol standardization efforts. Here we demonstrate that harmonization of ChIP-seq workflows across cell types and conditions is possible when obtaining chromatin from properly isolated nuclei. We established an ultrasound-based nuclei extraction method (NEXSON: Nuclei EXtraction by SONication) that is highly effective across various organisms, cell types and cell numbers. The described method has the potential to replace complex cell-type-specific, but largely ineffective, nuclei isolation protocols. By including NEXSON in ChIP-seq workflows, we completely eliminate the need for extensive optimization and sample-dependent adjustments. Apart from this significant simplification, our approach also provides the basis for a fully standardized ChIP-seq and yields highly reproducible transcription factor and histone modifications maps for a wide range of different cell types. Even small cell numbers (∼10,000 cells per ChIP) can be easily processed without application of modified chromatin or library preparation protocols.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Fractionation / methods
  • Cell Fractionation / standards*
  • Cell Line
  • Cell Nucleus / genetics
  • Cells, Cultured
  • Chromatin / isolation & purification
  • Chromatin Immunoprecipitation / methods*
  • Female
  • Hep G2 Cells
  • High-Throughput Nucleotide Sequencing / methods*
  • Histones / metabolism
  • Humans
  • Male
  • Mice
  • Reproducibility of Results
  • Sequence Analysis, DNA / methods*
  • Sonication
  • Transcription Factors / metabolism

Substances

  • Chromatin
  • Histones
  • Transcription Factors