Rnase r digestion

First, total RNA was extracted from tissue homogenate and cells using TRIzol (Life Technologies, Carlsbad, CA, United States). Then, total RNA was purified using the miRNeasy Mini kit (QIAGEN, Hilden, Germany) and assessed for purity using a NanoVue spectrophotometer (Ge BioSciences, United States). Total RNA, combined with anchored oligo (dT)18 primer and reverse transcriptase from the Transcriptor High FidelitycDNA Synthesis Kit (Roche, Germany), was subjected to the first strand synthesis for cDNA. The cDNA samples were examined by qRT-PCR using FastStart™ universal SYBR^® Green (Roche) for determination of miR-1296-5p and STAT1 mRNA, using U6 and beta-actin as the endogenous control. For hsa_circ_0086735, total RNA was prepared to the linear RNA using RNase R digestion (Epicentre Technologies, Madison, WI, United States). RNase R-treated RNA was reversely transcribed to cDNA using SuperScript III First Strand Synthesis System (Invitrogen). qRT-PCR was performed using Power Up Sybr Green Master Mix (Thermo Fisher Scientific). Each sample was assayed in triplicate determinations at least.

Total RNA isolated from HCC tissues using TRIzol reagent (Invitrogen, CA, USA) was assessed using Agilent 2100 Bioanalyzer pico-RNA chips (Agilent, CA, USA). During circRNA sequencing, DNase I (Epicenter) was used to remove DNA contamination. Ribosomal RNA was removed using an Epicenter Ribo-zero rRNA Removal Kit (Epicenter, USA), and RNase R digestion (Epicenter, USA) was subsequently performed to remove linear RNA. circRNA libraries were constructed with the NEBNext^® Ultra™ Directional RNA Library Prep Kit for Illumina^® (NEB, USA) following the manufacturer’s recommendations. The Illumina sequencing data were used to analyze the expression of circRNAs in tumors and adjacent tissues, using the limma package. Significantly differentially expressed circRNAs were defined as adjusted P < 0.05, and fold-change ≥ 2 or ≤0.5. A heatmap of the differentially expressed genes was generated using Cluster 3.0 software.