Improved Degradome Sequencing Protocol via Reagent Recycling from sRNAseq Library Preparations.

One 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. This work presents the first degradome-seq library preparation protocol that enables successful construction of libraries, even from highly degraded RNA samples with RIN below 3, thus significantly expanding the possibilities for research when working with low-quality material. The developed protocol improves the efficiency of library preparation in degradome-seq analysis used to identify miRNA targets, reduces library preparation time, and lowers the cost of purchasing reagents by using reagents from the RNA-seq library preparation kit and proprietary-designed primers. A crucial feature of this new protocol is optimizing the purification step for short library fragments, which increases the yield of correctly sized fragments compared to previously used methods. This is achieved by implementing an original method involving tube-spin purification with gauze and precipitation using sodium acetate with glycogen, greatly enhancing recovery efficiency-a factor especially critical when working with degraded RNA. Cloning to a plasmid and sequencing of the inserted fragment verified the correctness of the library preparation using the developed protocol. This protocol represents a groundbreaking tool for degradome research, enabling the construction and sequencing of degradome libraries, even from degraded samples previously considered unsuitable for such analyses. This is due to the use of residues from the sRNA-seq library kit. It noticeably reduces the cost of library construction. The precision of the excised fragment after electrophoresis was performed during the procedure to isolate fragments of the correct length, which was improved using additional size markers. Compared to previously used methods, optimizing the purification method of degradome-seq libraries allowed an increase in the yield of fragments obtained.