GSM7045804: HeLa siEIF4A3 YTHDF2-CLIP rep2; Homo sapiens; RIP-Seq
After irradiation, cells were harvested with a scraper, transferred to a microtube, pelleted at 4000 rpm for 3 min at 4°C, and then lysed in lysis buffer (150 mM NaCl, 0.5% NP-40, 50 mM Tris-HCl (pH 7.5), 2 mM EDTA) with cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail and SUPERase In at 4°C for 1 hour. Subsequently, the lysis mixture was centrifuged at 17,000 g at 4°C for 30 min and the supernatant was carefully collected. The samples were then treated with RNase T1 (1 u/µL) at RT for 15 min and centrifuged to collect the supernatant, 10% of which was saved as input. Then YTHDF2 antibody-conjugated protein G beads prepared by incubating antibody and beads at 4°C for 6 hours were added into the samples and incubate overnight. After beads washing, 10 u/µl RNase T1 was added and incubated at RT for 8 minutes. After incubation, the beads were extensively washed and then resuspended in 50 µL of SDS-PAGE loading buffer and heated at 95°C for 5 min. The RNA was finally extracted by cutting and recovering the band of YTHDF2-RNA complex from the SDS-PAGE gel. Before library preparation, end-repair by PNK and ATP was performed for the input and IP RNAs. The Library was prepared by NEBNext® Small RNA Library Prep kit following manufacturer's protocols.