Baksa Ivett, Szittya György
National Agricultural Research and Innovation Centre, Agricultural Biotechnology Institute, Epigenetics Group, Szent-Györgyi A. 4., Gödöllő, 2100, Hungary.
Methods Mol Biol. 2017;1640:113-128. doi: 10.1007/978-1-4939-7165-7_7.
The method described here enables the high-throughput identification of cleaved mRNA targets of ARGONAUTE/small RNA complexes. The protocol is based on a modified 5'-rapid amplification of cDNA ends combined with deep sequencing of 3' cleavage products of mRNAs. Poly(A) RNA is purified from the tissue of interest which is followed by a 5'-RNA adapter ligation. The ligated products are then reverse transcribed, amplified, and digested with MmeI. After gel separation, a 3' double-stranded DNA adapter is ligated to the fragments, which are then amplified and index labeled for the high-throughput sequencing of pooled degradome libraries. Sequencing datasets from pooled libraries can be analyzed with different bioinformatic approaches.
本文所述方法能够高通量鉴定AGO/小RNA复合物切割的mRNA靶点。该方案基于改良的5'-cDNA末端快速扩增，并结合mRNA 3'切割产物的深度测序。从感兴趣的组织中纯化poly(A) RNA，随后进行5'-RNA接头连接。然后将连接产物进行逆转录、扩增并用MmeI酶切。凝胶分离后，将3'双链DNA接头连接到片段上，然后进行扩增和索引标记，用于混合降解组文库的高通量测序。来自混合文库的测序数据集可用不同的生物信息学方法进行分析。
