Antral Follicle Gene Expression Analysis in Sheep Oocytes

To analyze the stage-specific gene expression at distinct antral follicle in sheep oocytes, the cDNA of 10 oocytes was synthesized using a reverse transcription system and amplified using the Single Cell Sequence Specific Amplification Kit (Vazyme, Nanjing, China; Cat#P621-01) according to the manufacturer’s instructions [85 (link)]. qPCR was conducted with the TB Green^® Premix Ex Taq™ II (Tli RNaseH Plus) (Cat#RR820L) on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). Ovis aries actin beta (β-actin) was used to normalize the data, and gene expression levels were calculated using the comparative cycle threshold (2^−ΔΔCt) method. The primers were obtained from Sangon Biotech (Shanghai, China), and all qPCR primer pairs are listed in Supplementary Table S17.