Tgem spectrophotometer

Total RNAs were separated from H1299 cells with Trizol reagent (Invitrogen) following the manufacturer's manual. The concentration and purity of total RNAs were evaluated by a TGem spectrophotometer (TIANGEN). RNA integrity was determined by agarose gel electrophoresis (EPS300, Shanghai Tianneng Technology Co., LTD, China). Next, RevertAid First Strand cDNA Synthesis Kit (Thermo#K1622) was implemented to synthesize cDNAs. qRT-PCR was implemented by employing FastStart Universal SYBR Green Master (Roche). The expression of each gene was assessed with the 2^−ΔΔCt method. Table S2 displayed primer sequences.

Total RNA was extracted from each sample using the RNAprepPure Plant Kit DP441 (Tiangen Biotech Co., Ltd, Beijing, China) according to the manufacturer’s instructions, followed by DNase I (Tiangen Biotech Co., Ltd.) treatment to eliminate DNA contamination. The purity and concentration of RNA samples were measured using a TGem spectrophotometer (Tiangen Biotech Co., Ltd.) and integrity was checked on agarose gel electrophoresis. RNA samples with a concentration higher than 200 ng/μL and a A260/A280 ratio between 1.9 and 2.1 were required for cDNA preparation. The reverse transcription kit (TaKaRa, Shiga, Japan) was used to reverse transcribe each sample of RNA into first-strand cDNA according to the user manual. Briefly, 10 µL of Master Mix was prepared, including 1 µg RNA, 2 µL 5 × gDNA Eraser Buffer, 1 µL gDNA Eraser, and a certain amount of RNase Free dH₂O. The Master Mix was then incubated at 42 °C for 2 min. After that, 1 µL PrimeScript RT Enzyme Mix I, 1 µL RT Primer Mix, 4 µL 5 × PrimeScript Buffer 2 (for Real Time) and 4 µL RNase Free dH₂O were added and then incubated at 37 °C for 15 min. Finally, the RT reaction was terminated by incubating at 85 °C for 5 s. The obtained cDNA was then diluted with 10 times RNase Free dH₂O to conduct qPCR.