Assessment of chromatin preparation methods to study post-translational modifications at a whole genome scale in chicken skeletal muscle

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 cross-linking is known to be relatively inefficient compared to other tissues. 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 a broad-peak histone mark using chicken P. major muscle tissue.Results: We compared the ChIP-seq data of two experiments performed either using cross-linked or native muscle chromatin. X-ChIP and N-ChIP protocols were adapted from previous studies. Both protocols performed equivalently until sequencing, producing high-quality Illumina reads (q30 > 93 %). Forty millions of reads were analyzed for both X-ChIP-seq and N-ChIP-seq experiments following ENCODE recommendations. Surprisingly, H3K27me3 X-ChIP-seq analysis led to the identification of only two thousands enriched regions compared to 14-16 thousands 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 regions of common enrichment identified in this study. Conclusions: Our findings suggest that the cross-linking preparation of muscle chromatin for ChIP-seq can compromise the analysis of histone marks, in particular when high-throughput sequencing is subsequently performed. We therefore recommend the use of N-ChIP-seq to study histone marks at the genome-wide level in skeletal muscle tissues.