GSM8553792: ctrl_IP_rep2; Homo sapiens; RIP-Seq
cells were lysed with eCLIP buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate), and RNA was fragmented using RNAse I. Then, samples were subjected for overnight incubation at 4C with corresponding beads-conjugated antibodies. RNA fragments 3' end were dephosphorylated with FastAP (Thermo) and PNK (NEB), followed by 3' barcoded linker ligation (T4 RNA ligase I, NEB). Then, samples were ran on protein gel for an hour on 150V, followed by overnight transfer on nitrocellulose membrane. protein kDa size and 75kDa above it were extracted for IP and size-matched input and extracted from membrane with proteinase K. the samples subjected to reverse transcription (SSIII Thermo Fisher), and subsequent ExoSAP-IP and EDTA, NaOH and HCl treatment for degradation of excess RT primers and rna template. Additional 3' end ligation and PCR amplification (Q5 2X mix NEB) generated the final library products.