GSM8658130: RBM24_IP-2; Homo sapiens; RIP-Seq
CAL27 cells were irradiated once for 400 mJ/cm2, and lysed in ice-cold wash buffer. Cells lysis was performed in cold Wash buffer (1× PBS, 0.5% SDS, 0.5% NP-40 and 0.5% sodium deoxycholate) supplemented with a 400 U/mL RNase inhibitor (Takara) and protease inhibitor cocktail (Bimake) and incubate on ice for 30 min. Clear cell lysate by centrifugation at 10,000 rpm for 10 min at 4°C. Add RQ I (Promega, 1U/μl) to a final concentration of 0.1 U/μl and incubate in a heat block for 30 min at 37°C. Immediately afterward, a stop solution was added to the lysates to quench DNase. The mixture was then vibrated vigorously and centrifuged at 13,000 x g at 4℃ for 15 min to remove cell debris. Then RNA digestion by MNase (Thermo Scientific) was performed. cDNA libraries were prepared with the KAPA RNA Hyper Prep Kit (KAPA, KK8541). According to the manufacturer's instructions, the libraries were constructed and used for 150 nt paired-end sequencing through the Illumina NovaSeq system.