Efficient library preparation for next-generation sequencing analysis of genome-wide epigenetic and transcriptional landscapes in embryonic stem cells

Methods Mol Biol. 2014;1150:3-20. doi: 10.1007/978-1-4939-0512-6_1.

Abstract

Gene expression in embryonic stem (ES) cells is regulated in part by a network of transcription factors, epigenetic regulators, and histone modifications that influence the underlying chromatin in a way that is conducive or repressive for transcription. Advances in next-generation sequencing technology have allowed for the genome-wide analysis of chromatin constituents and protein-DNA interactions at high resolution in ES cells and other stem cells. While many studies have surveyed genome-wide profiles of a few factors and expression changes at a fixed time point in undifferentiated ES cells, few have utilized an integrative approach to simultaneously survey protein-DNA interactions, histone modifications, and expression programs during ES cell self-renewal and differentiation. To identify transcriptional networks that regulate pluripotency and differentiation, it is important to generate high-quality genome-wide maps of transcription factors, chromatin factors, and histone modifications and to survey global gene expression profiles. Here, to interrogate genome-wide profiles of chromatin features and to survey global gene expression programs in ES cells, we describe protocols for efficient library construction for next-generation sequencing of ChIP-Seq and RNA-Seq samples.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Animals
  • Cell Culture Techniques
  • Cell Line
  • Chromatin / genetics
  • Chromatin Immunoprecipitation
  • Cloning, Molecular
  • DNA, Complementary / biosynthesis
  • DNA, Complementary / genetics
  • Embryonic Stem Cells / metabolism*
  • Epigenesis, Genetic / genetics*
  • Epigenomics / methods*
  • Gene Library*
  • High-Throughput Nucleotide Sequencing / methods*
  • Mice
  • Sequence Analysis, RNA
  • Sonication
  • Transcription, Genetic / genetics*

Substances

  • Chromatin
  • DNA, Complementary