ABSTRACT

The location of nucleosomes in chromatin significantly impacts many biological processes including DNA replication, repair, and gene expression. A number of techniques have been developed for mapping nucleosome locations in chromatin including MN-Seq (micrococcal nuclease digestion followed by next-generation sequencing), ATAC-Seq (Assay for Transposase-Accessible Chromatin followed by next-generation sequencing), and ChIP-Seq (chromatin immunoprecipitation and fragmentation followed by next-generation sequencing). All of these techniques have been successfully used, but each with its own limitations. Recently, New England Biolabs has marketed a new kit, the NEBNext Ultra II FS Library Prep kit, for preparing libraries for next-generation sequencing from purified genomic DNA. This kit is based on a novel proprietary DNA fragmentation procedure which appears to cleave DNA that is not bound by proteins. Because DNA is fragmented directly in the FS kit, we tested whether the kit might also be useful for mapping the location of nucleosomes in chromatin. Using simian virus 40 (SV40) chromatin isolated at different times in an infection, we have compared nucleosome mapping using the NEB FS kit (referred to as FS-Seq) to MN-Seq, ATAC-Seq, and ChIP-Seq. Mapping nucleosomes using FS-Seq generated nucleosome profiles similar to those generated by ATAC-Seq and ChIP-Seq in regulatory regions of the SV40 genome. We conclude that FS-Seq is a simple, robust, cost-effective procedure for mapping nucleosomes in SV40 chromatin that should be useful for other forms of chromatin as well. We also present evidence that FS-Seq may be useful for mapping transcription factors.

摘要

核小体在染色质中的位置显着影响许多生物过程，包括 DNA 复制、修复和基因表达。已经开发了许多技术来绘制染色质中的核小体位置，包括 MN-Seq（微球菌核酸酶消化，然后进行下一代测序）、ATAC-Seq（转座酶可及染色质分析，然后进行下一代测序）和 ChIP- Seq（染色质免疫沉淀和片段化，然后进行下一代测序）。所有这些技术都已成功使用，但每种技术都有其自身的局限性。最近，New England Biolabs 推出了一款新试剂盒 NEBNext Ultra II FS Library Prep 试剂盒，用于从纯化的基因组 DNA 中制备用于下一代测序的文库。该试剂盒基于一种新颖的专有 DNA 片段化程序，该程序似乎可以切割未与蛋白质结合的 DNA。由于 DNA 在 FS 试剂盒中直接片段化，我们测试了该试剂盒是否也可用于绘制染色质中核小体的位置图。使用在感染的不同时间分离的猿病毒 40 (SV40) 染色质，我们将使用 NEB FS 试剂盒（称为 FS-Seq）的核小体作图与 MN-Seq、ATAC-Seq 和 ChIP-Seq 进行了比较。使用 FS-Seq 映射核小体生成的核小体谱类似于 ATAC-Seq 和 ChIP-Seq 在 SV40 基因组的调节区域中生成的核小体谱。我们得出的结论是，FS-Seq 是一种简单、稳健、经济高效的方法，用于在 SV40 染色质中定位核小体，它也应该适用于其他形式的染色质。