Improved Degradome Sequencing Protocol via Reagent Recycling from sRNAseq Library Preparations

Abstract

BackgroundOne of the key elements in the analysis of gene expression and its post-translational regulation is miRNAs. Degradome-seq analyses are performed to analyze the cleavage of target RNAs in the transcriptome. In this work, an improved library preparation protocol for degradome sequencing is presented. The developed protocol improves the efficiency of library preparation in degradome-seq analysis used to identify microRNA targets, reduces the time of library preparation and lowers the cost of purchasing reagents.. ResultsThe aim of this study was the development of an efficient protocol for the construction of degradome sequencing libraries using residual reagents from the sRNA-seq library preparation kit. To this end, modified primers and adaptors were designed. The library purification step based on automated electrophoresis and high-resolution agarose was modified and optimized in the presented protocol. Size standards of 60 and 65 bp were developed. They were prepared for precise band excision from the gel. Cloning to plasmid and sequencing of the inserted fragment, i.e., a fragment from the degradome library, verified the correctness of the library preparation using the developed protocol. ConclusionThe developed protocol allowed the construction and sequencing of degradome libraries even from RNA samples with low RIN. It significantly reduces the cost of library construction. This is due to the use of residues from the sRNA-seq library kit. The precision of the excised fragment after electrophoresis performed during the procedure to isolate fragments of the correct length is significantly improved by the use of additional size markers. Compared to previously used methods, optimizing the purification method of degradom-seq libraries allowed to increase the yield of fragments obtained. Notably, the time required for the entire library preparation protocol does not exceed three days, also a significant time savings.