GSM5732817: IgG_Input_Reh; Homo sapiens; RIP-Seq
Cells were lysed using 1mL of eCLIP lysis mix and were subjected to two rounds of sonication for 4 minutes with 30 second ON/OFF at 75% amplitude. 5μg of pre-validated NUDT21 (Santa Cruz Biotechnology, sc-81109) antibody was then pre-coupled to 50ul of Anti-Rabbit IgG Dynabeads (ThermoFisher), added to lysate equivalent to 100μg of RNA, and then incubated overnight at 4°C. Prior to immunoprecipitation, 2% of the sample was taken as the paired input sample, with the remainder magnetically separated and washed with eCLIP high stringency wash buffers. IP and input samples were cut from the membrane at the relative band size to 75kDa above. RNA adapter ligation, IP-western, reverse transcription, DNA adapter ligation, and PCR amplification were performed according to the published single-end seCLIP protocol (Van Nostrand, E. L. et al. 2017)