A filtration-based method of preparing high-quality nuclei from cross-linked skeletal muscle for chromatin immunoprecipitation

Kazunari Nohara, Zheng Chen, Seung Hee Yoo

Research output: Contribution to journalArticle

Abstract

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

Original languageEnglish (US)
Article numbere56013
JournalJournal of Visualized Experiments
Volume2017
Issue number125
DOIs
StatePublished - Jul 6 2017

Fingerprint

Chromatin Immunoprecipitation
Chromatin
Muscle
Myofibrils
Skeletal Muscle
Tissue
Muscles
Contamination
DNA
Ultracentrifugation
Skeletal Muscle Fibers
Protein Binding
Anti-Idiotypic Antibodies
Antibodies
Assays
Genes
Proteins
Fibers
Processing

Keywords

  • Biochemistry
  • Chromatin immunoprecipitation
  • Circadian time
  • Cross-linking
  • Issue 125
  • Nuclei isolation
  • Sequential filtration
  • Skeletal muscle

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

A filtration-based method of preparing high-quality nuclei from cross-linked skeletal muscle for chromatin immunoprecipitation. / Nohara, Kazunari; Chen, Zheng; Yoo, Seung Hee.

In: Journal of Visualized Experiments, Vol. 2017, No. 125, e56013, 06.07.2017.

Research output: Contribution to journalArticle

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