Degradome Sequencing

CD Genomics is now able to provide degradome sequencing service to facilitate a more comprehensive insight into plant microRNA landscape. By using our service, you can detect the mRNA targets of the microRNA in a highly sensitive and accurate manner.

microRNAs (miRNAs) are a class of endogenous 20-24 nt non-coding RNAs produced by highly precise excision from stem-loop precursors, which are important regulators of gene expression at the transcriptional and post-transcriptional levels. The mature miRNA is recruited into a RNA-induced silencing complex (RISC) which degrades mRNA targets and suppresses their translation. The complementarity between a miRNA and its targeted mRNA determines whether miRNAs modulate gene expression by targeting mRNAs for cleavage. Plant miRNAs have been largely implicated in degradation of their RNA targets by slicing precisely between the 10th and 11th nucleotides (nt) from the 5’ end of miRNAs.

Next-generation sequencing and bioinformatics prediction provide effective methods for plant miRNA discovery and analysis. Degradome sequencing (Degradome-Seq), also referred to as parallel analysis of RNA ends sequencing (PARE-seq), is a modified version of 5'-Rapid Amplification of cDNA Ends (RACE) by utilizing advanced NGS technology. Degradome sequencing is an efficient tool to identify miRNA-guided cleaved sites in mRNAs, which allows directly identification of their prospective miRNA targets on a large scale. In this method, degraded capped mRNA is adapter-ligated and reverse-transcribed. Fragments are then Mmel-digested, purified, 3’-adapter-ligated, and PCR-amplified. Deep sequencing of the cDNA provides information about uncapped transcripts that undergo degradation. The workflow of our Degradome sequencing process is demonstrated in Figure 1.

Figure 1. Schematic workflow of small RNA sequencing process.

Sequencing Strategy and Recommended Depth

• Illumina HiSeq SE50
• 5-10 M reads

Data analysis

We have a team with the expert knowledge and computational resources to help you achieve your data analysis objectives. Our bioinformatics analysis pipeline includes mapping to reference, identification of rRNAs, tRNAs, snRNAs, snoRNAs, polyN and other non-coding RNAs, distribution analysis of degradation fragments on selected region of genome, identification of target mRNAs, statistical summarization of mRNA degradation sites, GO/KEGG analysis, and identification of degradation mRNA related microRNAs from miRBase.

Sample Requirements

1. Sample type: Total RNA without degradation or DNA contamination.
2. Starting amount of total RNA: ≥ 15 µg
3. Sample conc.: ≥ 100 ng/µl
4. Sample purity: OD260/280 = 1.8~2.2

Key Features and Advantages

• Novel library preparation technology. High efficiency and fast turnaround time.
• High throughput. Simultaneously sequences and analyses all the miRNA-cleaved fragments.
• High Accuracy. Eliminate the significant false-positive hits in miRNA target gene by using advanced bioinformatics approaches.
• Comprehensive data analysis. Identification of microRNA and targeted mRNA and their distributions.

By harness of Illumina next-generation sequencing technology and experienced technicians, our advanced Degradome-Seq is able to effectively identify miRNA cleavage sites from the degradome and accurately infer target genes of miRNA by leveraging the analytic power NGS data bioinformatics.