title: Transcriptomic-based identification of miR-125a novel targets in human hepatocarcinoma cells
design: Transcriptomic-based identification of miR-125a novel targets in human hepatocarcinoma cells
library_name: 8_HepG2_125a2_p
library_construction_protocol: Human liver cancer cell lines HepG2 and HuH-7 were cultured in RPMI 1640 and DMEM, respectively, containing 10% fetal bovine serum, 2 mM L-glutamine, 50 U/mL penicillin, and 100 mg/mL streptomycin. The day before transfection, cells were trypsinized and seeded in medium without antibiotics in 12-well plates. Transfections were then performed with cells at 80–90% of confluence by using 3 mL of Lipofectamine2000 (Thermo Fisher Scientific) for 1 mg of nucleic acids, as described by the manufacturer. In particular, miR-125a mimic or its control (Ctrl-miRNA) with unrelated sequence (all from Dharmacon) were transfected at 50 nM; 0.05 mg or 0.2 mg of reporter constructs were used for transfecting HuH-7 or HepG2 cells, respectively. The transfection mix was replaced with complete medium after 6 h, and the analyses were performed 48 h after transfection.  Total RNA was extracted from cell cultures using the miRNeasy mini kit (Qiagen, Hilden, Germany). The yield and quality of RNA were evaluated by NanoDrop One spectrophotometer (NanoDrop), Qubit 4 Fluorometer (Invitrogen) and by TapeStation 4200 (Agilent Technologies). Indexed libraries were prepared from 100 ng of each purified RNA sample using the Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero™ (Illumina), according to the manufacturer's instructions. Libraries were quantified with the TapeStation 4200 (Agilent Technologies) and Qubit fluorometer (Invitrogen Co.). Libraries were pooled such that each index-tagged sample was present in equimolar amount.