miRNA-Seq of Trachinotus blochii: liver exosome sample 03
The miRNA library construction was performed through the following optimized procedures: Initially, total RNA was size-fractionated by PAGE electrophoresis to specifically isolate small RNAs in the 18-30 nt range. The purified RNA fragments were then subjected to 3' adapter ligation using a 5'-adenylated and 3'-blocked single-stranded DNA adapter. Subsequently, UMI-containing RT primers were introduced into the system to hybridize with both the RNA-linked 3' adapters and excess free adapters. Following this, 5' adapters were ligated to the 5' ends of the resulting products from the previous step. First-strand cDNA synthesis was carried out through reverse transcription extension using the UMI-labeled RT primers. The adapter-ligated cDNA was then amplified by PCR with high-fidelity polymerase to increase yield. Post-amplification, the library fragments were size-selected by PAGE electrophoresis to obtain products in the 110-130 bp range.After quality control assessment, the final library preparation involved denaturation of the double-stranded PCR products and circularization through the addition of specific primers to generate single-stranded circular DNA molecules, ready for sequencing.