Circular structure confirmation
The circular structure of circ_0001722 was confirmed by RNase R treatment and Sanger sequencing by divergent primer PCR. For RNase R treatment, 3 μg total RNA extracted from OS tissues and cell lines were incubated with 20 U RNAse R (Epicentre Biotechnologies, USA) in a 10 μl volume at 37 °C for 45 min, followed by 70 °C for 10 min to deactivate the RNase R. The treated RNAs were used for RT-PCR. For Sanger sequencing, PCR products amplified by divergent primers of circ_0001722 were inserted into the T vector and sequenced by Tsingke Biotechnology (Beijing) Co., Ltd. The result was crosschecked with the back-splicing junction sites of circ_0001722 supplied by circBASE [17].