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Related Experiment Video

Updated: Jan 21, 2026

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing
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4C-seq from beginning to end: A detailed protocol for sample preparation and data analysis.

Peter H L Krijger1, Geert Geeven1, Valerio Bianchi1

  • 1Oncode Institute, Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands.

Methods (San Diego, Calif.)
|July 29, 2019
PubMed
Summary
This summary is machine-generated.

This study presents an improved protocol for 4C-seq, a method to map genome interactions. The optimized protocol and data pipeline enable high-resolution analysis of 3D genome organization and chromatin loops.

Keywords:
4C-seq4D nucleomeChromatin foldingChromosome conformation capture (3C)Genome architecture

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Area of Science:

  • Genomics
  • Molecular Biology
  • Epigenetics

Background:

  • Chromosome conformation capture (3C) methods analyze 3D genome organization by measuring DNA contact frequencies via proximity ligation.
  • 4C-seq (4C-sequencing) is a derivative of 3C, utilizing inverse PCR and next-generation sequencing to identify genomic regions interacting with a specific DNA locus.
  • Understanding genome folding is crucial for deciphering gene regulation and higher-order chromatin structures.

Purpose of the Study:

  • To present an optimized, step-by-step protocol for 4C-seq library preparation, including improved PCR and purification strategies.
  • To introduce a data processing pipeline for analyzing 4C-seq data from raw reads to genome browser-compatible files.
  • To facilitate high-resolution analysis and interpretation of 3D genome organization and DNA contacts.

Main Methods:

  • Development of an improved protocol for 4C-seq sample preparation and sequencing library generation.
  • Optimization of PCR amplification and 4C template purification steps.
  • Implementation of a data processing pipeline for analyzing multiplexed 4C-seq reads from FASTQ files.

Main Results:

  • The improved protocol yields high-resolution contact profiles from limited sequencing reads.
  • The data pipeline generates files suitable for visualization in genome browsers and statistical analysis, including peak calling.
  • Successful generation and analysis of 4C-seq datasets are demonstrated, enabling the study of regulatory and architectural chromatin loops.

Conclusions:

  • The presented optimized protocol and data pipeline significantly enhance the ease and resolution of 4C-seq experiments.
  • This resource empowers researchers to generate, visualize, and interpret high-resolution 3D genome organization data.
  • The findings facilitate the study of specific DNA contacts, chromatin loops, and domain structures within the nucleus.