Exred

Total RNA was isolated using TRIzol (Invitrogen) with DNase I treatment, and the extracted RNA was dissolved in 80 μL RNase-free distilled water and stored at −80°C. DNase I-treated RNA (∼5 μg) was treated for 30 min at 37°C with or without 3 U/μg of RNase R (Epicentre, Charlotte, NC, USA), and 1 μL RNase R-treated RNA was directly reverse transcribed using an MMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit with a random hexamer primer (Invitrogen) according to the manufacturer’s protocol. The cDNA was diluted 10-fold before qRT-PCR analysis.
qRT-PCR experiments were performed in duplicate (technical replicates) using 2 × RealStar Green Fast Mixture (GenStar, Beijing, China) and a LightCycler 480 Real-Time PCR System (Roche, Basel, Switzerland). PCR program settings were 95°C for 10 min (initial denaturation); 95°C for 15 s, and 60°C for 1 min, for 45 cycles. Ct values were obtained by the second derivative maximum method and analyzed by the 2^−ΔΔCt method.⁴⁹ (link) PCR products were visualized after electrophoresis in 2% ExRed-stained (Zomanbio, Beijing, China) agarose gel, and purified through the V-ELUTE Gel Mini Purification Kit (Zomanbio, Beijing, China). The pBLUE-T plasmid was constructed by the pBLUE-T fast cloning kit (Zomanbio, Beijing, China), and Sanger sequencing was performed.

Total genomic DNA was extracted from tissues by DNA digestion buffer incubation (50 mM Tris-HCl pH 8.0, 100 mM EDTA pH 8.0, 100 mM NaCl, 1% SDS, 0.5 mg/mL proteinase K) overnight at 65°C with gentle shaking, and then treated with 10 μg/mL RNase A (Zomanbio, Beijing, China) at 37°C. Total RNA was isolated using TRIzol (Invitrogen) with DNase I treatment. The cDNA library was total RNA directly reverse transcribed using an MMLV Reverse Transcriptase first-Strand cDNA Synthesis Kit with a random hexamer primer (Invitrogen). The negative control was total RNA not treated with MMLV Reverse Transcriptase.
PCR experiments were performed in duplicates using the TransStart FastPfu Fly polymerase (TransGen Biotech, Beijing, China). PCR program settings were 95°C for 5 min (initial denaturation); 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, for 45 cycles. PCR products were visualized after electrophoresis in 2% ExRed-stained (Zomanbio, Beijing, China) agarose gel, and purified through the V-ELUTE Gel Mini Purification Kit (Zomanbio, Beijing, China). The pBLUE-T plasmid was constructed using the pBLUE-T fast cloning kit (Zomanbio, Beijing, China), and Sanger sequencing was performed.