Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY, 14853, USA.
Plant Methods. 2012 Sep 20;8(1):41. doi: 10.1186/1746-4811-8-41.
Deep sequencing is a powerful tool for novel small RNA discovery. Illumina small RNA sequencing library preparation requires a pre-adenylated 3' end adapter containing a 5',5'-adenyl pyrophosphoryl moiety. In the absence of ATP, this adapter can be ligated to the 3' hydroxyl group of small RNA, while RNA self-ligation and concatenation are repressed. Pre-adenylated adapters are one of the most essential and costly components required for library preparation, and few are commercially available.
We demonstrate that DNA oligo with 5' phosphate and 3' amine groups can be enzymatically adenylated by T4 RNA ligase 1 to generate customized pre-adenylated adapters. We have constructed and sequenced a small RNA library for tomato (Solanum lycopersicum) using the T4 RNA ligase 1 adenylated adapter.
We provide an efficient and low-cost method for small RNA sequencing library preparation, which takes two days to complete and costs around $20 per library. This protocol has been tested in several plant species for small RNA sequencing including sweet potato, pepper, watermelon, and cowpea, and could be readily applied to any RNA samples.
深度测序是发现新的小 RNA 的强大工具。Illumina 小 RNA 测序文库制备需要带有 5'，5'-腺苷二磷酸基部分的预腺苷酸化 3' 端接头。在没有 ATP 的情况下，该接头可以连接到小 RNA 的 3' 羟基上，而 RNA 自身连接和串联则受到抑制。预腺苷酸化接头是文库制备所需的最基本和最昂贵的成分之一，而商业上可用的接头很少。
我们证明，带有 5' 磷酸和 3' 氨基的 DNA 寡核苷酸可以被 T4 RNA 连接酶 1 酶促腺苷酸化，生成定制的预腺苷酸化接头。我们已经使用 T4 RNA 连接酶 1 腺苷酸化接头构建并测序了番茄（Solanum lycopersicum）的小 RNA 文库。
我们提供了一种高效且低成本的小 RNA 测序文库制备方法，该方法需要两天时间完成，每个文库的成本约为 20 美元。该方案已在包括甘薯、辣椒、西瓜和豇豆在内的几种植物物种中用于小 RNA 测序，并可轻松应用于任何 RNA 样本。
