Qrt supermix 2

RNA was extracted using TRIzol reagent (9109, TaKaRa) according to the manufacturer's protocol. First, genomic DNA was removed using template 200 ng RNA mixed with 2 μl 4 × gDNA wiper mix (R123‐01, Vazyme) and nuclease free water up to 8 μl of the final reaction volume. This reaction mixture was incubated at 42°C for 2 min. Reverse transcription was then performed using the reaction tube mentioned above mixed with 2 μl of 5 × qRT SuperMix II (R123‐01, Vazyme) and nuclease free water up to 10 μl of the final reaction volume. This reaction mixture was incubated at 50°C for 15 min and then for 2 min at 85°C. Next, the RT products (cDNA) obtained in the previous step were used as the template for qPCR. The qPCR reactions were carried out using SYBR Green qPCR Master Mix (Q141‐02, Vazyme) containing 1 μl of cDNA in a 10 μl final volume reaction following the manufacturer's instructions in an Applied Biosystems qPCR System (StepOne, Thermo Fisher, USA) with the following cycling conditions: 5 min at 95°C followed by 30 cycles of 10 s at 95°C and 30 s at 60°C. The primers used to amplify circRNA and mRNA transcripts were synthesized by Invitrogen. The sequences of the primers are summarized in Table S3.