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JBP1-seq: A fast and efficient method for genome-wide profiling of 5hmC

机译：JBP1-seq：一种快速高效的5hmC全基因组谱分析方法

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摘要

We developed a novel approach, J-binding protein 1 sequencing (JBP1-seq), that combines the benefits of an improved recombinant JBP1 protein, Nextera-based library construction, and next-generation sequencing (NGS) for genome-wide profiling of 5-hydroxymethylcytosine (5hmC). Compared with the original JBP1, this new recombinant JBP1 was biotinylated in vivo and conjugated to magnetic beads via biotin-streptavidin interactions. These modifications allowed a more efficient and consistent pull-down of beta-glucosyl-5-hydroxymethylcytosine (beta-glu-5hmC), and sequence-ready libraries can be generated within 4.5 h from DNA inputs as low as 50 ng. 5hmC enrichment of human brain DNA using the new JBP1 resulted in over 25,000 peaks called, which is significantly higher than the 4003 peaks enriched using the old JBP1. Comparison of the technical duplicates and validations with other platforms indicated the results are reproducible and reliable. Thus, JBP1-seq provides a fast, efficient, and cost-effective method for accurate 5hmC genome-wide profiling. (C) 2014 Elsevier Inc. All rights reserved.
机译：我们开发了一种新颖的方法，即J结合蛋白1测序（JBP1-seq），该方法结合了改进的重组JBP1蛋白，基于Nextera的文库构建和下一代测序（NGS）的优势，可进行5个全基因组分析-羟甲基胞嘧啶（5hmC）。与原始JBP1相比，这种新的重组JBP1在体内进行了生物素化，并通过生物素-链霉亲和素相互作用与磁珠偶联。这些修饰使得β-葡萄糖基-5-羟甲基胞嘧啶（β-glu-5hmC）的提取更加有效，一致，并且可以在低至50 ng的DNA输入下4.5小时内生成可用于序列的文库。使用新的JBP1富集5hmC的人脑DNA产生了超过25,000个峰，这明显高于使用旧的JBP1富集的4003个峰。与其他平台进行技术重复和验证的比较表明，结果可重复且可靠。因此，JBP1-seq为准确的5hmC全基因组概况分析提供了一种快速，高效且经济高效的方法。 （C）2014 Elsevier Inc.保留所有权利。

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