Cfx96 real time pcr detection system

According to the manufacturer’s protocol, total RNA was isolated using Trizol reagent (Takara, Beijing, China), and the mRNA and circRNA cDNAs were synthesized with HiScript^® III first Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The qRT-PCR reactions were carried out in the Bio-Rad CFX96 Real-time PCR Detection System, using AceQ^® qPCR SYBR Green Master Mix (without ROX) (Vazyme, Nanjing, China). The program was set to be a two-step method, 95° for 5 s, 60° for 30 s, and 40 cycles. The gene expression results were analyzed using the 2^^−ΔΔCT method, and GAPDH was used as an endogenous control for circRNA and mRNA expression. For circRNA, we designed divergent primers. After PCR, Sanger sequencing was performed to determine the circular structure of circRNAs. The primer sequence information of the qRT-PCR experiment is shown in Supplementary Table S1.

Based on RT-PCR amplification methods, the sorted ILC subsets were reverse- transcribed to obtain cDNA. cDNAs were synthesized with a Single Cell Sequence Specific Amplification Kit (Vazyme, Nanjing, China). First, the amplification primers of different genes were mixed to make an Assay Pool (the final concentration of each primer was 0.1 μM), and then the reaction system was configured in the Nuclease-free centrifuge tube as follows: 2x Reaction Mix 2.5ul, 0.1 uM Assay Pool 0.5ul, RT/Taq enzyme 0.1ul, Nuclease-free ddH20 up to 5.0 μl. Single-cell ILC subsets were added to the reaction system, and then the following reaction was performed on the PCR instrument: 50° for 60 min and 1 cycle, 95° for 3 min and 1 cycle, 95° for 15 s, 60° for 15 min and 14 cycles. At the end of the reaction, 20 μl of Nuclease-free ddH2O (1:5 dilution) was added to each tube, centrifuged at 3000 rpm (1000 × g) for 2 min, and subsequent qPCR reactions were performed immediately.
The qRT-PCR reactions were carried out in the Bio-Rad CFX96 Real-time PCR Detection System, using AceQ^® qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). The program was set as a two-step method, 95° for 10 s, 60° for 30 s and 40 cycles. The gene expression results were analyzed using the 2^−ΔΔCT method, and GAPDH was used as an endogenous control for mRNA expression.