Ab126741

To extract whole‐cell proteins, cells were lysed in 1 × RIPA buffer (Solarbio, Beijing, China) containing a cocktail solution of protease inhibitors (Sangon Biotech) on ice for 30 minutes and then centrifuged (12 000 × g for 10 minutes at 4°C). Subsequently, the Bradford method was used to normalize protein concentrations, and a representative Western blotting protocol was adopted. In brief, 20 μg of protein per channel was separated through 10% SDS‐PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with a blocking solution (5% skim milk in Tris‐buffered saline with 0.1% Tween‐20, TBS‐T) for 1 hour, the membranes were incubated with the primary antibodies (including rabbit anti‐human NR5A1: ab168380, GATA4: ab134057, DMRT1: ab126741, GAPDH: ab37168, Abcam, Cambridge, UK, at 1:2000; and rabbit anti‐human CYP17A1: orb213833, HSD3B1: orb5478, STAR:orb129747, CYP11A1: orb213832, Biorbyt, San Francisco, CA, USA, at 1:1000) overnight at 4°C. Next, the membranes were incubated with the goat anti‐rabbit secondary antibody (ab150079, Abcam, 1:10000) for 1 hour at RT in the dark, and specific bands were detected using a two‐colour Infrared Laser Imaging System (LI‐COR Odyssey, USA).

C&R for DMRT1 and normal rabbit IgG was performed as described^{43 (link)–45 (link)}. Briefly, 50,000 purified DZ⁺PGCLCs were washed and bound to activated 10 μl Concanavalin A-coated magnetic beads. The beads were then incubated with wash buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM spermidine and protease inhibitor) containing 0.1% digitonin and 1 μg of DMRT1 antibody (ab126741, Abcam) or normal rabbit IgG (#2729, Cell signaling) for 2 h at 4 °C on a rotator. After two washes in digitonin–wash buffer, beads were resuspended in Protein A/G-MNase fusion protein at 70 ng ml⁻¹ in digitonin–wash buffer and incubated for 1 h at 4 °C on a rotator. After two washes in digitonin–wash buffer (the beads with replicate 3 of day 4 DZ⁺PGCLC and day 8 DZ⁺PGCLC were washed with low-salt rinse buffer (20 mM HEPES, pH 7.5, 0.5 mM spermidine and 0.1% digitonin) once additionally), beads were resuspended in ice-cold calcium incubation buffer (3.5 mM HEPES pH 7.5, 10 mM CaCl₂ and 0.1% digitonin). After 15 min, 2× stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM egtazic acid, 0.1% digitonin, RNase A 100 μl ml⁻¹ and glycogen 50 μg ml⁻¹) was added. Beads were incubated at 37 °C for 30 min, the liquid was removed to a fresh tube and DNA was extracted with phenol–chloroform extraction.