An Assessment of Fixed and Native Chromatin Preparation Methods to Study Histone Post-Translational Modifications at a Whole Genome Scale in Skeletal Muscle Tissue

Background: 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.

Results: 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.

Conclusions: 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.