Gene Expression Analysis of Follicles

A TRIzol reagent (Invitrogen, Carlsbad, CA) was used to extract total RNA from follicles. The cDNA was generated from 2 μg total RNA using a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China), following the manufacturer's protocol. The qRT-PCR was performed using a SYBR® Premix Ex Taq™ Kit (Takara, DRR420A, Kyoto, Japan) on an ABI 7500HT Real-Time PCR Detection System (Applied Biosystems, Foster City, USA), with the following conditions: 95°C for 10 min and then 40 cycles of 95°C for 30 s, 64°C for 34 s, and 72°C for 30 s. Comparisons of expression levels were determined by the 2^−ΔΔCt formula method normalized to β-actin. The sequences for primers are listed in Table 1. The RNA-seq was carried out according to a previous study [25 (link)].