GSM6626515: N2A, basal, rep1; Mus musculus; RIP-Seq
When the cultures reached 80% confluency, media were replaced with PBS. The cells were crosslinked once at 400 mJ/cm2 and after that twice at 200 mJ/cm2 by using Stratagene UV Stratalinker 1800 (Stratagene, LaJolla, CA, USA). After crosslinking the cells were scraped off the plates and collected into tubes, pelleted, and stored at -80°C as a dry pellet until further processing. Each sample corresponded to approximately 60 million cells. The frozen samples after crosslinking were lysed and they went through Ago immunoprecipitation and radiolabeling of RNA. Ago-RNA complexes were further purified by SDS-PAGE and nitrocellulose transfer and visualized by autoradiography. Products were then extracted from the membrane, amplified with PCR, analyzed with Bioanalyzer and sequenced (Illumina).