An improved method of constructing degradome library suitable for sequencing using Illumina platform

Yong-Fang Li; Miao Zhao; Menglei Wang; Junqiang Guo; Li Wang; Jie Ji; Zongbo Qiu; Yun Zheng; Ramanjulu Sunkar

doi:10.1186/s13007-019-0524-7

An improved method of constructing degradome library suitable for sequencing using Illumina platform

Plant Methods. 2019 Nov 18:15:134. doi: 10.1186/s13007-019-0524-7. eCollection 2019.

Authors

Yong-Fang Li¹, Miao Zhao¹, Menglei Wang¹, Junqiang Guo², Li Wang¹, Jie Ji¹, Zongbo Qiu¹, Yun Zheng^{2

3}, Ramanjulu Sunkar⁴

Affiliations

¹ 1College of Life Sciences, Henan Normal University, Xinxiang, Henan People's Republic of China.
² 2Faculty of Information Engineering and Automation, Kunming University of Science and Technology, Kunming, 650500 Yunnan China.
³ 3Yunnan Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, 650500 China.
⁴ 4Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078 USA.

Abstract

Background: Post-transcriptional gene regulation is one of the critical layers of overall gene expression programs and microRNAs (miRNAs) play an indispensable role in this process by guiding cleavage on the messenger RNA targets. The transcriptome-wide cleavages on the target transcripts can be identified by analyzing the degradome or PARE or GMUCT libraries. However, high-throughput sequencing of PARE or degradome libraries using Illumina platform, a widely used platform, is not so straightforward. Moreover, the currently used degradome or PARE methods utilize MmeI restriction site in the 5' RNA adapter and the resulting fragments are only 20-nt long, which often poses difficulty in distinguishing between the members of the same target gene family or distinguishing miRNA biogenesis intermediates from the primary miRNA transcripts belonging to the same miRNA family. Consequently, developing a method which can generate longer fragments from the PARE or degradome libraries which can also be sequenced easily using Illumina platform is ideal.

Results: In this protocol, 3' end of the 5'RNA adaptor of TruSeq small RNA library is modified by introducing EcoP15I recognition site. Correspondingly, the double-strand DNA (dsDNA) adaptor sequence is also modified to suit with the ends generated by the restriction enzyme EcoP15I. These modifications allow amplification of the degradome library by primer pairs used for small RNA library preparation, thus amenable for sequencing using Illumina platform, like small RNA library.

Conclusions: Degradome library generated using this improved protocol can be sequenced easily using Illumina platform, and the resulting tag length is ~ 27-nt, which is longer than the MmeI generated fragment (20-nt) that can facilitate better accuracy in validating target transcripts belonging to the same gene family or distinguishing miRNA biogenesis intermediates of the same miRNA family. Furthermore, this improved method allows pooling and sequencing degradome libraries and small RNA libraries simultaneously using Illumina platform.

Keywords: Cleavage; Degradome; Illumina sequencing; Target gene; miRNA.