title: GSM8853873: AGS, RBM8A, rep2; Homo sapiens; RIP-Seq
library_construction_protocol: Cell lysis: AGS cells were washed twice with ice-cold PBS and then lysed in RIP lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) supplemented with protease inhibitors and RNase inhibitors on ice for 30 minutes. The cell lysate was centrifuged at 12,000 × g for 10 minutes at 4 °C to remove cell debris. RNA immunoprecipitation: The supernatant was pre-cleared with protein A/G magnetic beads for 1 hour at 4 °C. Then, the pre-cleared lysate was incubated with anti - RBM8A antibody overnight at 4 °C with gentle rotation. After that, protein A/G magnetic beads were added and incubated for another 2 hours at 4 °C. The beads were washed 5 times with RIP wash buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) to remove non - specifically bound proteins and RNAs. RNA extraction: The RNA bound to the beads was extracted using TRIzol reagent according to the manufacturer's instructions. The extracted RNA was treated with DNase I to remove genomic DNA contamination and then purified using an RNA purification kit. RIP-seq