High-throughput identification of gene regulatory sequences using next-generation sequencing of circular chromosome conformation capture (4C-seq)

Erin A. Brettmann, Inez Y. Oh, Cristina de Guzman Strong

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

Original languageEnglish
Article numbere58030
JournalJournal of Visualized Experiments
Volume2018
Issue number140
DOIs
StatePublished - Jan 1 2018

Keywords

  • Bait
  • Chromatin
  • Enhancers
  • Genetics
  • Issue 140
  • Looping
  • Lysis
  • Regulatory
  • Restriction enzyme
  • Sequencing

Fingerprint Dive into the research topics of 'High-throughput identification of gene regulatory sequences using next-generation sequencing of circular chromosome conformation capture (4C-seq)'. Together they form a unique fingerprint.

  • Cite this