Agilent feature extraction software version 11.0.1.1

Total RNA from each sample was quantified using a NanoDrop ND-1000. The sample preparation and microarray hybridization were performed based on Arraystar's standard protocols (KANGCHEN Biotech, Shanghai, China). In brief, total RNA was digested with RNase R (Epicentre, Inc.) to remove linear RNAs and enrich for circular RNAs. Then, the enriched circRNAs were amplified and transcribed into fluorescent cRNA using a random priming method (Arraystar Super RNA Labeling Kit; Arraystar). The labeled cRNAs were hybridized onto the Arraystar Human circRNA Array V2 (8×15K, Arraystar). Next, the slides were washed and the arrays were scanned using an Agilent Scanner G2505C. Agilent Feature Extraction software (version 11.0.1.1) was applied to analyze the acquired array images. Quantile normalization and subsequent data processing were performed using the R software limma package. Differentially expressed circRNAs with statistical significance between two groups were identified through Volcano Plot filtering. Significantly differentially expressed circRNAs were then further filtered through analysis of Fold Change. Hierarchical Clustering was performed to highlight distinguishable circRNAs expression patterns among samples.

The panel of cell lines (three biological replicates for each) were profiled using the Arraystar Human circRNA Array version 2.0 (Arraystar, MD, United States). The sample preparation and microarray hybridization were performed according to manufacturer’s instructions. Briefly, total RNA was digested with RNAse R (Epicenter, Illumina, CA, United States) to remove linear RNAs and enrich for circRNAs. The enriched circRNAs were amplified and transcribed into fluorescent cRNA using a random priming method with Arraystar Super RNA Labeling Kit (Arraystar). The labeled cRNAs were hybridized onto the Arraystar Human circRNA Array V2 (8 × 15 K). The array slides were washed and scanned on the Agilent Scanner G2505C. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images.

The paired tumor and adjacent normal tissue samples from six dCCA patients were used for microarray detection. Total RNA from each sample was extracted with TRIzol reagent (Invitrogen) and quantified using the NanoDrop ND-1000 (Thermo Fisher, USA). The products were digested with Rnase R (Epicentre, Madison, WI, USA), and the linear RNAs were removed. The enriched circRNAs were amplified and transcribed into fluorescent cRNA utilizing a random primer method (Super RNA Labeling Kit, Arraystar, Rockville, MD, USA). Labeled cRNAs were hybridized onto the Arraystar Human circRNA Array V2 (8x15K) and incubated for 17 h at 65° C in a hybridization oven (Agilent, Santa Clara, CA, USA). After washing the slides, the arrays were scanned using an Agilent G2505C scanner.
Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization of the raw data was performed using R software. CircRNA samples with at least three out of six having flags in “P” or “M” (“All Targets Value”) were retained for further analyses. The limma R package was applied to identify DE circRNAs between tumor and normal tissues (|log₂FC| > 1 and p < 0.05). We used pheatmap and Rcircos R packages to visualize the DE circRNAs and their distributions in human chromosomes.