Gel-seq: whole-genome and transcriptome sequencing by simultaneous low-input DNA and RNA library preparation using semi-permeable hydrogel barriers.
Gel-seq: whole-genome and transcriptome sequencing by simultaneous low-input DNA and RNA library preparation using semi-permeable hydrogel barriers.
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DOI：
10.1039/c7lc00430c
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发表时间：
2017-07
期刊：
影响因子：
6.1
通讯作者：
G. Hoople;Andrew Richards;Yan Wu;Kota Kaneko;Xiaolin Luo;G. Feng;Kun Zhang;A. Pisano
中科院分区：
文献类型：
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作者：
G. Hoople;Andrew Richards;Yan Wu;Kota Kaneko;Xiaolin Luo;G. Feng;Kun Zhang;A. Pisano
下一代测序技术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测特定样本的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，使研究人员能够同时从同一样品中测序DNA和RNA。这项技术可以直接检测基因组变化对100个细胞中转录组的影响。Gel-seq协议的核心是DNA与RNA的物理分离。这种分离是通过使用一种新设计的装置来实现的，该装置包含几种不同的聚丙烯酰胺膜。在这里，我们报告的发展和验证该设备。我们提出了该设备的制造协议和用于制备基因文库的生物协议。使用具有均匀表达的细胞系（PC 3和Hela），我们表明用Gel-seq生成的文库与使用RNA或DNA的标准方法开发的文库相似。此外，我们通过从小鼠肝肿瘤收集的100个细胞样本中生成匹配的基因组和转录组文库来证明Gel-seq的能力。
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.