4.4. miRNA Library Construction and Sequencing

The miRNA sequencing library was constructed using the QIAseq miRNA Library Kit (QIAGEN, Germany, Cat. No. 331502). In brief, the miRNA sequencing library was prepared by the following steps: (1). 3′-adaptor ligation with pre-adenylated adaptor; (2). 5′-adaptor ligation with sequenced primers; (3). cDNA synthesis using reverse transcription primers with a unique molecular index (UMI) assigned to every miRNA molecule, allowing the identification of individual molecules; (4). cDNA cleanup; (5). PCR amplification using primers with sample barcode; (6). library cleanup. The quality of the library was checked using the 5200 Fragment Analyzer System (Agilent Technologies, CA, USA). The size of the library product is between 190 and 220 bps. The library was quantified by Qubit (Thermo Fisher Scientific, USA, Cat. No. {"type":"entrez-protein","attrs":{"text":"Q32851","term_id":"75280859","term_text":"Q32851"}}Q32851), and the concentration must be more than 1 ng/μL for the following sequencing assay. The library was sequenced on the Illumina NextSeq 550 (Illumina, CA, USA) as per the manufacturer’s instructions.