GSM8776106: healthy control 2; Homo sapiens; miRNA-Seq
Total RNA was extracted using the Total RNA Extraction Kit (Thermo Fisher Scientific Inc., MA, USA), following the manufacturer's instructions. RNA quantity and quality were determined using the Nanodrop 2000 (Thermo Fisher Scientific, MA, USA) and Agilent 2100 Bioanalyzer systems (Agilent Technologies). Only samples with an RNA Integrity Number (RIN) above 7 were utilized to construct a high-quality total RNA sequencing (RNA-seq) library. For library preparation, 1 μg of total RNA or 10 ng of small RNA was used. The 3' SR Adaptor for Illumina was attached to the small RNA using a 3' ligation enzyme. To prevent adaptor-dimer formation, excess 3' SR Adaptor was hybridized with the SR RT Primer for Illumina. The 5' SR Adaptor for Illumina was then attached to the small RNA using a 5' ligation enzyme, and first-strand cDNA was synthesized using ProtoScript II Reverse Transcriptase. Each sample was amplified by PCR using P5 and P7 primers, and the PCR products were purified using DNA clean beads. The purified products, ranging from 140 to 160 bp, were recovered and cleaned using PAGE, and validated with an Agilent 2100 Bioanalyzer. Libraries with different indexes were multiplexed, and the qualified libraries were sequenced on the Illumina MGI2000 System (MGI Tech Co., Ltd, China) using paired-end PE150.