title: GSM8152215: Ion_018; Homo sapiens; miRNA-Seq
library_construction_protocol: Fresh tissues were fragmented with a scalpel and homogenised using the Fisherbrand™ 150 handheld homogenizer (Fisherbrand™). RNA was extracted from 25 mg of fresh tissue using the mirVana miRNA Isolation Kit (Ambion), following manufacturer's recommendations. The RNA integrity of the extracted total RNA was analysed using the Agilent RNA 6000 Nano Kit on an Agilent 2100 Bioanalyzer (Agilent Technologies) and assessed by the RNA integrity number (RIN). A RIN cut-off value of ≥6.0 was considered for subsequent miRNA library preparation. Library preparation was performed using the CleanTag Small RNA Library Preparation Kit (TriLink Biotechnologies®) following manufacturer's protocol. Briefly, 100 ng of the isolated total RNA were employed and sequential steps of 3' and 5' adapter ligation, reverse transcription (RT) reaction of tagged RNA library and PCR amplification were performed from each RNA sample. PCR products were purified following a 2-step protocol with the Agencourt AMPure XP Beads (Beckman Coulter™), yielding a total of 15 μL of purified library. Then, library preparation efficacy was evaluated using the Agilent DNA 1000 Kit on an Agilent 2100 Bioanalyzer (Agilent Technologies). A 142pb peak, representing tagged small RNA of ~21nt in the small RNA libraries, was considered suitable. Finally, miRNA sequencing was performed on an Ion GeneStudio™ S5 Systems (Ion Torrent™), using an Ion 540™ Chip (Ion Torrent™) with appropriate reagents.