To profile the whole miRNome, sRNA-seq was performed on liver tumor and nodular tissue of SRF- mice alongside the corresponding controls. The library was generated using TruSeq Small RNA Library Prep Kits v2 (Illumina) according to the manufacturer’s protocol. Subsequent to identification of tumor-associated miRNAs, we performed screening of potential miRNA targets using the DIANA microT-CDS (17 (link)) and TargetScan (18 (link)) databases. To increase the accuracy of down-regulated miRNA target predictions, we performed RNA-seq analyses on largely overlapping samples as used for sRNA-seq.
To quantify mature miRNA, pre-miRNA, and pri-miRNA expression in RNA samples of fibrosis models, total RNA was reverse transcribed using the miScript II RT Kit (Qiagen) and the miScript SYBR Green PCR Kit (Qiagen) was used to quantify the RNA expressions.
To experimentally validate the functionality of predicted miRNA targeting, a luciferase gene reporter assay was used.
To validate PPAR binding to miRNA promoters, the ChIP protocol based on the procedure described by Daniel et al. (47 (link)) was used with some modifications.
Detailed description of all animal models, materials, and methods is contained inSI Appendix .
To quantify mature miRNA, pre-miRNA, and pri-miRNA expression in RNA samples of fibrosis models, total RNA was reverse transcribed using the miScript II RT Kit (Qiagen) and the miScript SYBR Green PCR Kit (Qiagen) was used to quantify the RNA expressions.
To experimentally validate the functionality of predicted miRNA targeting, a luciferase gene reporter assay was used.
To validate PPAR binding to miRNA promoters, the ChIP protocol based on the procedure described by Daniel et al. (47 (link)) was used with some modifications.
Detailed description of all animal models, materials, and methods is contained in