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].