4C-seq from beginning to end: A detailed protocol for sample preparation and data analysis

Peter H L Krijger, Geert Geeven, Valerio Bianchi, Catharina R E Hilvering, Wouter de Laat*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

44 Citations (Web of Science)

Abstract

Chromosome conformation capture (3C) methods measure DNA contact frequencies based on nuclear proximity ligation, to uncover in vivo genomic folding patterns. 4C-seq is a derivative 3C method, designed to search the genome for sequences contacting a selected genomic site of interest. 4C-seq employs inverse PCR and next generation sequencing to amplify, identify and quantify its proximity ligated DNA fragments. It generates high-resolution contact profiles for selected genomic sites based on limited amounts of sequencing reads. 4C-seq can be used to study multiple aspects of genome organization. It primarily serves to identify specific long-range DNA contacts between individual regulatory DNA modules, forming for example regulatory chromatin loops between enhancers and promoters, or architectural chromatin loops between cohesin- and CTCF- associated domain boundaries. Additionally, 4C-seq contact profiles can reveal the contours of contact domains and can identify the structural domains that co-occupy the same nuclear compartment. Here, we present an improved step-by-step protocol for sample preparation and the generation of 4C-seq sequencing libraries, including an optimized PCR and 4C template purification strategy. In addition, a data processing pipeline is provided which processes multiplexed 4C-seq reads directly from FASTQ files and generates files compatible with standard genome browsers for visualization and further statistical analysis of the data such as peak calling using peakC. The protocols and the pipeline presented should readily allow anyone to generate, visualize and interpret their own high resolution 4C contact datasets.

Original languageEnglish
Pages (from-to)17-32
Number of pages16
JournalMethods
Volume170
Early online date26 Jul 2019
DOIs
Publication statusPublished - 1 Jan 2020
Externally publishedYes

Keywords

  • 4C-seq
  • Chromosome conformation capture (3C)
  • Chromatin folding
  • Genome architecture
  • 4D nucleome
  • CHROMATIN DOMAINS
  • HIGH-RESOLUTION
  • HUMAN GENOME
  • PRINCIPLES
  • ORGANIZATION
  • ALIGNMENT
  • SINGLE

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