title: GSM9428023: Primary mouse granulosa cells, Veh treatment,IgG, rep1_SMAD2tagged; Mus musculus; OTHER
library_construction_protocol: For CUT&RUN experiments, approximately 55,000 cells were used for each reaction. Concanavalin A-coated magnetic beads (Bangs Laboratories, BP531) were conjugated to the cells, and the resulting bead–cell complexes were resuspended in 100 ul of Antibody Buffer per reaction. Immunoprecipitation was initiated by adding 1 ug of the indicated antibodies: IgG control (Sigma, I5006), anti-HA (EpiCypher, 13-2010), anti-PA (Fujifilm, NZ-1), or anti-SMAD2 (Abcam, ab33875). After overnight incubation at 4 C, the bead-cell complexes were washed and subsequently incubated with pAG-MNase (EpiCypher, 15-1016) at room temperature for 10 minutes. bead-cell-pAG-MNase complexes were washed and chromatin digestion was initiated by adding 1 ul of 100 mM CaCl2 to each reaction, followed by incubation at 4 C for 2 hours. Reactions were terminated by adding 50 ul of Stop Buffer and incubating at 37 C for 10 minutes. The supernatant was collected, and DNA was purified by phenol–chloroform extraction and ethanol precipitation. For RNA experiments, total RNA from mouse granulosa cells was extracted using QIAGEN RNeasy kit (QIAGEN, Cat 74104) following the manufacturer's protocol. For Cut and Run experiments, sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (New England BioLabs, E7645) according to the manufacturer's instructions, and paired-end 150 bp sequencing was performed. For RNA-seq experiments, NEBNext Ultra II Directional RNA Library Prep Kit (NEB, E7760S, E7490S) with 500 ng input RNA following the manufacturer's protocol.