Rnase r

RNA extraction from tissues and cells was done using the miRNeasy Mini Kit as per the manufacturing protocols (Qiagen). RNA with a purity of 1.96–2.04 (A260/280) was used for further experiments.
RNase R treatment was carried out by adding 10 U RNase R (Geneseed) into a 20 µl reaction containing 3 µg of total RNA, followed by RNA purification with phenol–chloroform extraction after 20 min incubation at 37°C.

Total RNA was extracted using the RNA-Quick Purification Kit (Vazyme, Shanghai, China). For the RNase R treatment, 2 μg of total RNA was incubated with or without 3 U/mg RNase R (Geneseed, Guangzhou, China) for 15 min at 37°C. For the Actinomycin D treatment, transcription was prevented by the addition of 2 mg/ml Actinomycin D. One μg of total RNA was transcribed into cDNA on an ABI Veriti™ 96-Well Thermal Cycler (Thermo Fisher) using HiScript II Q RT SuperMix for qPCR (R223-01, Vazyme) for mRNA and circRNA analysis, and miRNA 1st Strand cDNA Synthesis Kit (MR101-01, Vazyme) for miRNA analysis. The qRT-PCR reaction was carried out in a Thermofisher 7500 Real-Time PCR system using AceQ Universal SYBR qPCR Master Mix for mRNA and circRNA (Q511-02, Vazyme) and miRNA Universal SYBR qPCR Master Mix (MQ101-01/02, Vazyme). All sequences were presented in Supplementary Table S1.

Total RNAs was extracted by TRIzol reagent (Invitrogen) according to the manufacturer's protocol, and subsequently treated with DNase I (Promega) at 37 ºC for 30 minutes, followed by 95 ºC for in-activation of DNase I. To digest linear RNA, 1 μg of total RNAs from 293T cells was subjected to 4 U RNase R (Geneseed) treatment in 20 μl reactions at 37 ºC for 20 minutes, followed by RNase R heat inactivation at 70 ºC for 10 min. 1 μg of RNA was applied to synthesize cDNA using Reverse Transcriptase (Takara) according to the manufacturer's instructions. The RT products were then diluted 1:20 used for PCR amplification. Semi-quantitative PCR was performed by incubation at 94 °C for 5 min followed by 25 cycles of 94 °C for 30 s, 61 °C for 30 s, and 72 °C for 10 s on a T100 PCR Thermal Cycler (Bio-Rad). RT-PCR products were then separated on 2.5% agarose gels running with 1× TAE buffer, and scanned with a scanner (Tanon, 2500R). The resulting bands intensity was quantified by Image J software. Real-time PCR was performed using Premix Pro Taq HS qPCR kit (AG11701). ΔΔCt (Cycle threshold) was calculated using housekeeping gene GAPDH. Statistical analysis was performed using a Student's t-test. The primers used in the semi-quantitative PCR and qPCR were listed in Table S1.