P2051

As described previously [70 (link)], RIP was performed using 16.5 dpc mouse ovaries. The ovaries were lysed in cell lysis buffer (P0013, Beyotime, Shanghai, China) containing PMSF (1:100, ST506, Beyotime, Shanghai, China), a proteinase inhibitor cocktail (1:100, P1005, Beyotime, Shanghai, China), DTT (1:50, ST041-2 ml, Beyotime, Shanghai, China), and an RNase inhibitor (1:20, R0102-10 kU, Beyotime, Shanghai, China). After incubation on ice for 20 min, the lysate was added to 20 μl of protein A agarose (P2051-2 ml, Beyotime, Shanghai, China) for preclearing at 4 °C for 1 h. Two micrograms of an SRSF1 antibody (sc-33652, Santa Cruz Biotechnology, California, USA) and a normal mouse IgG (sc-3879, Santa Cruz Biotechnology, California, USA) were added to the lysate, followed by overnight incubation at 4 °C. The next day, 60 μl of protein A agarose was added to the lysate followed by incubation at 4 °C for 4 h. The agarose complexes containing antibodies, target proteins, and RNA were washed for 5 min at 4 °C, which was repeated 3 times. Protein-bound RNA was then extracted using RNAiso Plus and the Direct-zol RNA MicroPrep kit. RIP–qPCR was performed according to the above RT–qPCR protocol.

As described previously (Gagliardi and Matarazzo, 2016 (link)), RIP was performed using 5 dpp mouse testes. The testes were lysed in cell lysis buffer (P0013, Beyotime) containing PMSF (1:100, ST506, Beyotime), a proteinase inhibitor cocktail (1:100, P1005, Beyotime), DTT (1:50, ST041-2 ml, Beyotime), and an RNase inhibitor (1:20, R0102-10 kU, Beyotime). After incubation on ice for 20 min, the lysate was added to 20 μl of protein A agarose (P2051-2 ml, Beyotime) for pre-clearing at 4°C for 1 hr. Two micrograms of an SRSF1 antibody (sc-33652, Santa Cruz Biotechnology) and a normal mouse IgG (sc-3879, Santa Cruz Biotechnology) were added to the lysate, which was then incubated overnight at 4°C. The next day, 60 μl of protein A agarose was added to the lysate, and the mixture was incubated at 4°C for 4 hr. The agarose complexes containing antibodies, target proteins, and RNA were washed three times for 5 min at 4°C and repeated. Protein-bound RNA was extracted using RNAiso Plus and a Direct-zol RNA MicroPrep Kit. RIP-qPCR was performed according to the above RT-qPCR protocol.