Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Heidelberg, Germany.
Nat Protoc. 2017 Jan;12(1):122-149. doi: 10.1038/nprot.2016.163. Epub 2016 Dec 15.
Here we describe a protocol for NAD captureSeq that allows for the identification of nicotinamide-adenine dinucleotide (NAD)-capped RNA sequences in total RNA samples from different organisms. NAD-capped RNA is first chemo-enzymatically biotinylated with high efficiency, permitting selective capture on streptavidin beads. Then, a highly efficient library preparation protocol tailored to immobilized, 5'-modified RNA is applied, with adaptor ligation to the RNA's 3' terminus and reverse transcription (RT) performed on-bead. Then, cDNA is released into solution, tailed, ligated to a second adaptor and PCR-amplified. After next-generation sequencing (NGS) of the DNA library, enriched sequences are identified by comparison with a control sample in which the first step of chemo-enzymatic biotinylation is omitted. Because the downstream protocol does not necessarily rely on NAD-modified but on 'clickable' or biotin-modified RNA, it can be applied to other RNA modifications or RNA-biomolecule interactions. The central part of this protocol can be completed in ∼7 d, excluding preparatory steps, sequencing and bioinformatic analysis.
在这里，我们描述了一种 NAD captureSeq 方案，该方案可用于鉴定来自不同生物体的总 RNA 样品中的烟酰胺腺嘌呤二核苷酸 (NAD)-帽 RNA 序列。NAD-帽 RNA 首先通过高效的化学酶法进行生物素标记，从而能够在链霉亲和素珠上进行选择性捕获。然后，应用一种专门针对固定化、5'-修饰 RNA 的高效文库制备方案，在 RNA 的 3'末端连接衔接子，并在珠上进行反转录 (RT)。然后，cDNA 释放到溶液中，加尾、连接到第二个衔接子并进行 PCR 扩增。对 DNA 文库进行下一代测序 (NGS) 后，通过与省略了化学酶法生物素化第一步的对照样品进行比较，鉴定富集的序列。由于下游方案不一定依赖于 NAD 修饰，而是依赖于“点击化学”或生物素修饰的 RNA，因此它可以应用于其他 RNA 修饰或 RNA-生物分子相互作用。该方案的核心部分可在约 7 天内完成，不包括预备步骤、测序和生物信息学分析。
