Researchers from the National Agricultural Research and Innovation Centre, Hungary describe a method that 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.
A schematic representation of degradome library preparation
After the sequence-specific miRNA-induced RNA cleavage, the 3′ cleavage product of the mRNA contains a 5′-monophosphate and a poly(A) tail. The method starts with poly(A) selection and followed by a 5′-RNA adapter ligation. The next step is reverse transcription to make the first strand of cDNA with a 3′-adapter oligo (dT) primer. A few PCR cycle amplifications of the template and a control step are carried out before the MmeI digestion. This type IIS restriction enzyme cleaves 20 nucleotides, 3′ away from the recognition site. The cleaved fragment is ligated to a 3′-dsDNA adapter , and a PCR amplification with an index primer labeling is done. The PAGE-purified product is ready for deep sequencing.