An improved method of constructing degradome library suitable for Illumina high-throughput sequencing platform

Post-transcriptional gene regulation is a critical layer of overall gene expression programs and microRNAs (miRNAs) play an indispensable role in this process by guiding cleavage on the messenger RNA targets. The miRNA-guided cleavage on the mRNA targets can be confirmed by analyzing the sequenced degradome or PARE or GMUCT libraries. However, high-throughput sequencing of PARE or degradome libraries is not as straightforward as sequencing small RNA libraries. Moreover, the currently used degradome or PARE methods utilize Mme1 restriction site and the resulting fragments are only 20-nt long, which often poses difficulty in distinguishing between the family members of the same gene family. In this modified degradome protocol, EcoP15I recognition site is introduced to the 3' end of the 5’RNA adaptor of TruSeq small RNA library, the double strand DNA adaptor sequence is modified to suit with the ends generated by the EcoP15I. These modifications allow amplification of the degradome library by primer pairs used for small RNA library preparation. Therefore, degradome library generated using this protocol can be sequenced as easily as small RNA library, and the resulting tag length is ~27-nt, which is longer than previous methods (20-nt). The protocol allows sequencing small RNA and degradome libraries simultaneously. Examination of degraded polyA RNA with a monophospahate at 5’end. These RNA is ligated to an RNA adapter consisting Ecop15I recognition site at 3’end. After ligation and RT-PCR, the PCR product was digested with Ecop15I and then ligated to a double-stranded DNA oligo. The final degradome library is enriched by primer pairs used for small RNA library preparation. Therefore, the generated degradome library can be sequenced as easily as small RNA library using Illumina sequencing platform.