RNA-seq of human primary ovarian granulosa cells with knockdown of ESR2 isoform 1, treated with estradiol
Five women undergoing in vitro fertilization treatment for infertility due to tubal or male factors at Antoine Béclère Hospital (Clamart, France) were included in this study. All patients underwent a standard gonadotropin-releasing hormone (GnRH) antagonist stimulation protocol. Follicular fluid containing granulosa cells was collected 36 h after administration of recombinant human chorionic gonadotropin via transvaginal ultrasound-guided aspiration. Informed written consent was obtained from all participants. Ethical approval was granted by the institutional review board (IRB Blefco-IORG0010582) under registration number “2021-1”. After oocyte retrieval, granulosa lutein cells (hGCs) were purified from the follicular fluid. Briefly, follicular fluid was centrifuged through a one-step Percoll gradient at 4,000 × g for 15 min to remove red blood cells. hGCs were collected at the interface, washed with DPBS, resuspended in growth medium, and cultured at 37°C in a humidified incubator with 5% CO₂.  Viral infection and E2 treatment of primary cultures of granulosa cells: Primary cultured hGCs were seeded in 12-well plates at 0.6 × 106 cells per well in growing medium and maintained at 37°C for 24 h. Cells were then transduced with viral particles (ESR2-V1 (B1) targeting or scramble (S)) at a multiplicity of infection (MOI) of 90 viral particles per cell (optimized titer) in 250 µL of DMEM/F12 medium without phenol red. Three hours post-infection, treatment medium containing 2 µg/mL puromycin was added to ensure selection of successfully transduced cells. Twenty-four hours after transduction, cells were treated for an additional 24 hours with either vehicle (CT) or 10 nM E2 in fresh treatment medium containing puromycin.    Total RNA was extracted from each condition using the RNeasy® Micro Kit   Extracted RNA samples underwent quality control (QC) using the Agilent Bioanalyzer 2100 and were quantified with a Qubit Fluorometer (Thermo Fisher Scientific). cDNA library preparation and RNA sequencing were carried out by Novogene (Cambridge, UK). Libraries were prepared from 200 ng of input RNA and PCR-amplified indexed 