4.11. Chromatin Accessibility Assay

The chromatin accessibility assay in this study was conducted using the EpiQuik™ Chromatin Accessibility Assay Kit (EPIGENTEK, Farmingdale, NY, USA) following the manufacturer’s instructions. Granulosa cells (GCs) were collected and suspended in 1 × lysis buffer. The cell suspension was divided into sample and No-Nse control groups. Both groups were incubated on ice, which was followed by vortexing and centrifugation to remove the supernatant. The chromatin was washed with 1 mL 1 × wash buffer at 4 °C and centrifuged to discard the supernatant. Subsequently, NDB and Nse were added to the sample group, while the No-Nse control group was treated with NDB only. The Nse reaction mixture was added, which was followed by incubation of the chromatin pellet under specified conditions. DNA was eluted from the binding columns using elution solution (ES) and centrifugation. We designed three primer pairs in the promoter region of SLOC3A1 (region-1, 282 bp, +971/+1252 bp, region-2, 125 bp, +1433/+1557 bp, and region-3, 209 bp, +1623/+1831 bp, transcription start site = +2000, Table 5). Hieff ^® qPCR SYBR Green Master Mix and a CFX96 Touch Real-Time PCR system were used to measure the amplification efficiency. The reaction volume was consistent with that of the RT-qPCR reaction. Amplification programs were performed using a two-step method: pre-denaturation at 95 °C for 10 min, which was followed by 40 cycles including denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. The fold enrichment (FE) in the SLCO3A1 promoter region was calculated using the formula FE = 2^{(Nse CT − no-Nse CT)} × 100%.

Primers used for the chromatin accessibility of the SLCO3A1 promoter region.