Chamq universal sybr green master mix

RNA was extracted using the Trizol reagent according to the manufacturer’s instructions, and complementary DNA (cDNA) was reversed transcribed from RNA with the HiScript II Q RT SuperMix for qPCR kit (Vazyme, China). qRT-PCR was performed using ChamQ Universal SYBR Green Master Mix (Vazyme, China) on QuantStudioTM Dx system (Life Technologies) and the relative expression levels of the mRNA were calculated by 2^−ΔΔCt method and normalized to β-actin. The primers used for qRT-PCR are listed below:: human LDHB F: 5’- CCTCAGATCGTCAAGTACAGTCC-3’; human LDHB R: 5’-ATCACGCGGTGTTTGGGTAAT-3’; mouse LDHB F: 5’-CATTGCGTCCGTTGCAGATG-3’; mouse LDHB R: 5’- GGAGGAACAAGCTCCCGTG-3’; DNMT1 F: 5’- CCTAGCCCCAGGATTACAAGG-3’; DNMT1 R: 5’-ACTCATCCGATTTGGCTCTTTC-3’; DNMT3A F: 5’-CCGATGCTGGGGACAAGAAT-3’; DNMT3A R: 5’-CCCGTCATCCACCAAGACAC-3’; DNMT3B F: 5’-CCCAGCTCTTACCTTACCATCG-3’; DNMT3B R:5’- GGTCCCCTATTCCAAACTCCT-3’. Experiments were performed at least twice.

To validate the reliability of circRNAs, we designed divergent primers encompassing back-splice junctions for five candidate DE-circRNAs (Supplementary File). All circRNA primers were run on an agarose gel and were sequence-validated. The reverse transcription of circRNA was performed using HiScript II 1st Strand cDNA Synthesis kit (Vazyme, Nanjing, China). Next, real-time qPCR was performed with ChamQ Universal SYBR Green Master Mix (Vazyme, Nanjing, China) in QuantStudio Real-Time PCR Systems (ThermoFisher, Foster City, CA, USA), to validate the expression of the selected DE-circRNAs. In addition, six candidate IncRNAs were also validated by RT-qPCR. Data were quantified using the 2^{−ΔΔ CT} method, and normalized to the internal reference gene U6.