Small RNA seq of HepG2 cells expressing shRNA targeting wild-type, mutated or both ß-catenin versus shRNA control
Small RNA seq of HepG2 cells expressing shRNA targeting wild-type, mutated or both ß-catenin versus shRNA control
Cells were treated with trypsin, centrifuged and washed in PBS. After centrifugation, the cell pellet was frozen at -80°C until the RNA extraction process. HepG2 cell line were maintained at 37°C in a humidified 5% CO2 atmosphere in Dulbecco's modified Eagle's medium (DMEM: 4.5 g/l glucose, glutamax, pyruvate) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Total RNA extraction was performed using the Direct-zol RNA Microprep kit (Zymo Research, R2062). SmallRNA-seq libraries were prepared using the QIAseq miRNA Library kit (QIAGEN, 331502) and QIAseq miRNA 96 index Kit IL UDI-F (QIAGEN, 331665) according to manufacturer's protocol. Briefly, undiluted 3' adapters were first ligated on 100ng of total RNA. Then, undiluted 5' adapters were ligated and RNA retro-transcribed with undiluted RT primers to generate double strand cDNA. A final PCR of 16 cycles was performed to amplify libraries and barcode samples with unique dual indices. An additional purification step allowed to obtain 200-300 bp fragments.