Shiley-Marcos School of Engineering, University of San Diego, 5998 Alcala Park, San Diego, CA 92110, USA.
Lab Chip. 2017 Jul 25;17(15):2619-2630. doi: 10.1039/c7lc00430c.
The advent of next generation sequencing has fundamentally changed genomics research. Unfortunately, standard protocols for sequencing the genome and the transcriptome are incompatible. This forces researchers to choose between examining either the DNA or the RNA for a particular sample. Here we describe a new device and method, collectively dubbed Gel-seq, that enables researchers to simultaneously sequence both DNA and RNA from the same sample. This technology makes it possible to directly examine the ways that changes in the genome impact the transcriptome in as few as 100 cells. The heart of the Gel-seq protocol is the physical separation of DNA from RNA. This separation is achieved electrophoretically using a newly designed device that contains several different polyacrylamide membranes. Here we report on the development and validation of this device. We present both the manufacturing protocol for the device and the biological protocol for preparing genetic libraries. Using cell lines with uniform expression (PC3 and Hela), we show that the libraries generated with Gel-seq are similar to those developed using standard methods for either RNA or DNA. Furthermore, we demonstrate the power of Gel-seq by generating a matched genome and transcriptome library from a sample of 100 cells collected from a mouse liver tumor.
下一代测序的出现从根本上改变了基因组学研究。不幸的是，用于测序基因组和转录组的标准方案是不兼容的。这迫使研究人员在检查特定样本的 DNA 或 RNA 之间做出选择。在这里，我们描述了一种新的设备和方法，统称为 Gel-seq，它使研究人员能够从同一个样本中同时对 DNA 和 RNA 进行测序。这项技术使得直接研究基因组变化对转录组的影响成为可能，其样本中细胞数量少至 100 个。Gel-seq 方案的核心是 DNA 与 RNA 的物理分离。这是通过使用新设计的设备电泳来实现的，该设备包含几个不同的聚丙烯酰胺膜。我们在此报告该设备的开发和验证。我们介绍了设备的制造方案以及用于制备遗传文库的生物学方案。使用具有均匀表达的细胞系（PC3 和 Hela），我们表明，使用 Gel-seq 生成的文库与使用标准方法分别生成的 RNA 或 DNA 文库相似。此外，我们通过从小鼠肝肿瘤中收集的 100 个细胞的样本生成匹配的基因组和转录组文库，展示了 Gel-seq 的强大功能。
