Hoople Gordon D, Richards Andrew, Wu Yan, Pisano Albert P, Zhang Kun
Department of Engineering, University of San Diego;
Department of Bioengineering, University of California, San Diego.
J Vis Exp. 2018 Mar 26(133):57315. doi: 10.3791/57315.
The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.
从小量起始样本中扩增并测序DNA或RNA的能力仅仅是在过去五年才得以实现。不幸的是，用于生成基因组或转录组文库的标准方案并不兼容，研究人员必须针对特定样本选择对DNA还是RNA进行测序。凝胶测序（Gel-seq）通过使用一个简单的水凝胶装置，使研究人员能够从100 - 1000个细胞开始，同时为DNA和RNA制备文库，从而解决了这个问题。本文介绍了该装置的详细制作方法以及生成配对文库的生物学方案。我们设计的凝胶测序方法易于其他研究人员实施；许多遗传学实验室已经拥有复制凝胶测序装置所需的设备。我们的方案采用了常用于全转录本扩增（WTA）和文库制备的试剂盒，对于那些已经熟悉生成基因组和转录组文库的研究人员来说也可能很熟悉。我们的方法使研究人员能够在不进行样本分割的情况下，以可忽略不计的额外成本，将DNA和RNA测序的能力应用于单个样本。
