GSM7094519: IgG2; Mus musculus; RIP-Seq
Total cells were isolated from adult WT C57BL/6N mouse testes, and then the cells were cross-linked by ultraviolet light (254 nm) to maintain the covalent binding of RBPs to their cognate RNA. Subsequently, SRSF1 and cross-linked RNAs were immunoprecipitated with an anti-SRSF1 antibody and digested with micrococcal nuclease (EN0181, Thermo Fisher Scientific). An IR800-biotin adapter was ligated to the 3′ ends of the RNA fragments. Then, the SRSF1/RNA complexes were separated by SDS‒PAGE and transferred to a nitrocellulose membrane (HATF00010, Millipore). These RNA and protein complexes from approximately 47 to 62 kDa were extracted from the nitrocellulose membrane, after which proteinase K (9034, Takara) digestion was performed. RNA was isolated with saturated phenol (AM9712, Ambion), RNA ligated with adaptors, and converted to cDNA with a SuperScript III First-Strand Kit (18080-051, Invitrogen). The cDNA was amplified by PCR to prepare the corresponding libraries and then sequenced on illumina NovaSeq 6000.