Rnase r

Total RNA (2 µg) was isolated and subjected to incubation at 37 °C for one hour in both the presence and absence of RNase R (5 U/µg; Beyotime, Shanghai, China). Subsequent qRT-PCR analysis allowed us to determine levels of circFTO and FTO mRNA.

Total RNA was subjected to 2 distinct procedures: one portion was treated with RNase R (4 U/μg) (Beyotime) and incubated for 20 min. The other portion was kept as a control and was not subjected to any treatment. After these treatments, the processes of reverse transcription and RT-qPCR were executed following the procedures outlined in the RNA extraction and RT-qPCR section.

S/P fractionation was performed as previously described (39 ). About 48 h after transfection, the HEK 293T or HeLa cells were harvested and lysed in 100 μl of a RIPA buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and cocktail protease inhibitor (Roche)) on ice for 30 min, and then the lysates were centrifuged at 13,000 rpm for 15 min at 4 °C. The 90-μl supernatant was added with 30 μl of 4× loading buffer (8% SDS), while the pellet was sufficiently washed with the RIPA buffer three times at 4 °C and then added with 60 μl of 4× loading buffer (8% SDS). Equal volumes of the supernatant and pellet fractions were subjected to SDS-PAGE and Western blotting analysis. When needed, RNase A (Invitrogen) with a final concentration of 0.5 μg/μl or RNase R (Beyotime Biotechnology) with a final unit of 0.5 U/μl was included in the lysis buffer for digesting RNA.

RNA was extracted from the collected cells and digested with RNase R (10 U/μL, Beyotime, Shanghai, China) for 15 min. Then cDNA was synthesized, and RT-qPCR analysis was performed to verify the stability of circ-LIMK1.

Total RNA (2 µg) was incubated with RNase R (20 U/µL, Lucigen, Wisconsin, United States) for 20 min at 37°C. Control samples were exposed to the same experimental steps as those used for the experimental samples, except for the addition of RNase R. The qRT-PCR products were added to 6 loading buffer (Beyotime) with the nucleic acid dye Gel-Green (Beyotime). After agarose gel (2%) electrophoresis at 60 V for 40 min, DNA fragments were visualized using UV transillumination. Moreover, qRT-PCR amplification products were excised from the agarose gel and purified using a GeneJET Gel Purification Kit (Thermo Fisher Scientific). Subsequently, following the standard approach detailed by Geneseed (Guangzhou, China), the nucleotide sequences of the purified products were determined via Sanger sequencing.

The cyclization of circ_8521 was validated using RNase R (Beyotime, Shanghai, China) by following the manufacturer’s instructions. Briefly, ~4 µg of isolated RNA was incubated with RNase R (3 U/µg RNA) for 30 min at 37 °C, which was subsequently deactivated at 70 ℃ for 10 min. Finally, quantitative real-time (qRT)-PCR analysis was performed.