A native chromatin purification system for epigenomic profiling in C. elegans

We describe an in vivo chromatin purification system for genome-wide epigenetic profiling in C. elegans. In this system, we coexpressed the E. coli biotin ligase enzyme (BirA), together with the C. elegans H3.3 gene fused to BioTag, a 23-amino acid peptide serving as a biotinylation substrate for BirA, in vivo in worms. We developed methods to isolate chromatin under different salt extraction conditions, followed by affinity purification of biotinylated chromatin with streptavidin and genome-wide profiling with microarrays. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf All experiments were done using two channels per chip, comparing DNAs extracted from either salt-extracted or insoluble chromatin to whole nuclear chromatin, whole nuclear chromatin to randomly fragmented genomic DNA, streptavidin-bound biotin-tagged histone-variant-containing chromatin to salt-extracted chromatin, gel-purified mononucleosomes to whole EDTA-extracted soluble chromatin, or streptavidin-bound biotin-tagged histone-variant-containing chromatin to whole EDTA-extracted soluble chromatin to randomly fragmented DNA from embryo nuceli.

本研究描述了一种用于秀丽隐杆线虫（C. elegans）全基因组表观遗传谱分析的活体内染色质纯化系统。在该系统中，我们在秀丽隐杆线虫体内共表达了大肠杆菌生物素连接酶（BirA），以及融合有BioTag的秀丽隐杆线虫H3.3基因；BioTag是一段可作为BirA生物素化底物的23氨基酸肽段。我们开发了在不同盐抽提条件下分离染色质的方法，随后通过链霉亲和素亲和纯化生物素标记的染色质，并利用微阵列芯片开展全基因组谱分析。有关数据使用的条款与条件，请参阅http://www.genome.gov/27528022 及 http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf。所有实验均采用每芯片双通道设计，具体比较组别如下：盐抽提染色质或不溶性染色质的提取DNA与全细胞核染色质；全细胞核染色质与随机片段化基因组DNA；结合链霉亲和素的生物素标记组蛋白变体染色质与盐抽提染色质；凝胶纯化的单核小体与EDTA抽提的全可溶性染色质；以及结合链霉亲和素的生物素标记组蛋白变体染色质、EDTA抽提的全可溶性染色质与随机片段化的胚胎细胞核基因组DNA。