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.

中文翻译：

---

固定和天然染色质制备方法的评估，以研究骨骼肌组织中全基因组规模的组蛋白翻译后修饰。

背景技术通常通过染色质的免疫沉淀，然后进行定量PCR（ChIP-qPCR）或高通量测序（ChIP-seq）来分析与组蛋白标记相关的基因组基因座。染色质可以是交联的（X-ChIP）或以天然状态使用（N-ChIP）。DNA和蛋白质的交联有助于在分析之前稳定它们之间的相互作用。尽管X-ChIP是最常用的方法，但已知肌肉组织固定效率相对较低。而且，没有方案描述简单且可靠的骨骼肌染色质的制备，其质量足以用于随后的高通量测序。在这里，我们旨在建立和比较两种染色质制备方法，用于使用鸡主要体育组织对H3K27me3（宽峰组蛋白标记）进行全基因组分析。结果从鸡的肌肉组织（胸大肌）制备了固定和未固定的染色质。染色质固定，通过超声或消化剪切和免疫沉淀均等效。获得了高质量的Illumina读数（q30> 93％）。使用epic（基于SICER的工具）和MACS2对数据进行生物信息学分析。X-ChIP-seq和N-ChIP-seq实验均分析了四千万个读数。出乎意料的是，与在N-ChIP-seq情况下鉴定的约15,000个区域相比，H3K27me3 X-ChIP-seq分析仅鉴定了2000个富集区域。与X-ChIP-seq相比，重复之间的N-ChIP-seq峰更一致。通过ChIP-qPCR在已知在肌管中富集H3K27me3的PAX5和SOX2基因座以及本研究中确定的常见富集基因座处，通过ChIP-qPCR证实了更高的N-ChIP-seq富集。结论我们的发现表明，在交联条件下为ChIP-seq制备肌肉染色质可能会损害对宽组蛋白标记的系统分析。因此，当必须在骨骼肌组织上进行ChIP实验时，尤其是考虑到广泛的信号源时，天然染色质制剂应优先于交联。