Determination of mt-RNA Termini by cRT-PCR

cRT-PCR was used to determine the termini of mt-RNAs as previously described (Supplemental Fig. S9; Yokobori and Paabo 1995 (link); Forner et al. 2007 ). Briefly, 5 µg of mt-RNA from one sample was allowed to self-ligate in a total volume of 100 µL containing 100 units of T4 RNA ligase (Takara) at 16°C for 16–18 h. The circular RNAs were purified using the PureLink RNA Mini Kit (Invitrogen). RT-PCR was performed as previously described (Wang et al. 2017 (link)). Primer sequences are provided in Supplemental Table S1. For all RT-PCR experiments, a negative control without template RNA was included. PCR products were cloned into the TA vector using the TAcloning kit (Takara) according to the manufacturer's protocol and sequenced (Sangon Technology).