An assessment of fixed and native chromatin preparation methods to study histone post-translational modifications at a whole genome scale in skeletal muscle tissue - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Biological Procedures Online Année : 2017

An assessment of fixed and native chromatin preparation methods to study histone post-translational modifications at a whole genome scale in skeletal muscle tissue

Sabine Crochet
  • Fonction : Auteur
  • PersonId : 964844
Thierry Bordeau
  • Fonction : Auteur
  • PersonId : 1204849
Nathalie Couroussé
  • Fonction : Auteur
  • PersonId : 969508
Aurélien Brionne
Anne Collin
Vincent Coustham

Résumé

Genomic loci associated with histone marks are typically analyzed by immunoprecipitation of the chromatin followed by quantitative-PCR (ChIP-qPCR) or high throughput sequencing (ChIP-seq). Chromatin can be either cross-linked (X-ChIP) or used in the native state (N-ChIP). Cross-linking of DNA and proteins helps stabilizing their interactions before analysis. Despite X-ChIP is the most commonly used method, muscle tissue fixation is known to be relatively inefficient. Moreover, no protocol described a simple and reliable preparation of skeletal muscle chromatin of sufficient quality for subsequent high-throughput sequencing. Here we aimed to set-up and compare both chromatin preparation methods for a genome-wide analysis of H3K27me3, a broad-peak histone mark, using chicken P. major muscle tissue. Fixed and unfixed chromatin were prepared from chicken muscle tissues (Pectoralis major). Chromatin fixation, shearing by sonication or digestion and immunoprecipitation performed equivalently. High-quality Illumina reads were obtained (q30 > 93%). The bioinformatic analysis of the data was performed using epic, a tool based on SICER, and MACS2. Forty millions of reads were analyzed for both X-ChIP-seq and N-ChIP-seq experiments. Surprisingly, H3K27me3 X-ChIP-seq analysis led to the identification of only 2000 enriched regions compared to about 15,000 regions identified in the case of N-ChIP-seq. N-ChIP-seq peaks were more consistent between replicates compared to X-ChIP-seq. Higher N-ChIP-seq enrichments were confirmed by ChIP-qPCR at the PAX5 and SOX2 loci known to be enriched for H3K27me3 in myotubes and at the loci of common regions of enrichment identified in this study. Our findings suggest that the preparation of muscle chromatin for ChIP-seq in cross-linked conditions can compromise the systematic analysis of broad histone marks. Therefore, native chromatin preparation should be preferred to cross-linking when a ChIP experiment has to be performed on skeletal muscle tissue, particularly when a broad source signal is considered.
Fichier principal
Vignette du fichier
2017_David_ReproducticeProceduresOnline_1.pdf (2.34 Mo) Télécharger le fichier
2017_David_BiologicalProceduresOnline_2.pdf (2.34 Mo) Télécharger le fichier
Origine : Fichiers éditeurs autorisés sur une archive ouverte
Origine : Fichiers éditeurs autorisés sur une archive ouverte
Loading...

Dates et versions

hal-01608235 , version 1 (26-05-2020)

Licence

Paternité - Partage selon les Conditions Initiales

Identifiants

Citer

Sarah-Anne David, Benoit Piegu, Christelle Hennequet-Antier, Maëlle Pannetier, Tiphaine Aguirre-Lavin, et al.. An assessment of fixed and native chromatin preparation methods to study histone post-translational modifications at a whole genome scale in skeletal muscle tissue. Biological Procedures Online, 2017, 19, pp.1-11. ⟨10.1186/s12575-017-0059-0⟩. ⟨hal-01608235⟩
115 Consultations
49 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More