Escherichia coli poly a polymerase

The in vitro polyadenylation was carried out using Escherichia coli poly(A) polymerase (Invitrogen) as described in (26 (link)).

Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Reverse transcription of 2 μg total RNA was performed using the EasyScript First-Strand cDNA Synthesis SuperMix (Transgen Biotech, Beijing, China). For the microRNA analysis, a poly(A) tail was added to total RNA (0.5 μg) by Escherichia coli poly(A) Polymerase (Invitrogen), oligo(dT) adapter primers, and M-MLV Reverse Transcriptase (Invitrogen) for reverse transcription. RT-qPCR was performed using the 7500 Fast Real-Time PCR System (ThermoFisher Scientific, Waltham, MA) and Go Taq qPCR Master Mix (Promega, Madison, WI). The cycling conditions for RT‑qPCR were as follows: 95°C for 5 min, followed by 45 cycles of 95°C for 10 s, 55°C for 30 s, and 72°C for 30 s. Relative expression levels of miR-421, circ_0000567, and TMEM100 were normalized to those of endogenous U6, GAPDH, and β-actin, respectively. The fold changes in transcript levels were calculated using the 2^-ΔCt method. All primers are listed in Table S1.