GSM5835427: URB1_iCLIP_rep2; Homo sapiens; RIP-Seq
HeLa cells expressing Flag-tagged URB1 were UV-crosslinked, resuspended in lysis buffer and sonicated for 20 s x 6 times on 25% duty. Supernatants were incubated with anti-Flag antibodies (Shanghai Genomics) or control IgG-conjugated beads for 2h at 4°C. Beads with protein-RNA complexes were treated with 5 Kunz U/ml of micrococcal nuclease in the presence of 5 μg yeast tRNAs for 5 min at 37°C, followed by dephosphorylation of RNA 3' ′ ends with PNK. Then pre-adenylated linker L3 (3′ modification with biotin) was linked to 3′ end of RNA using T4 RNA ligase 1 at 16°C overnight. The 5′ end of RNA was labeled with [γ- 32P] ATP using PNK. Protein-RNA complexes were eluted with 1x Nupage loading buffer (Invitrogen) at 70°C for 10 min, resolved on 4%–12% NuPAGE Bis-Tris gel (Invitrogen), transferred to a nitrocellulose membrane and exposed to a Fuji film at −80°C. Bands corresponding to specific protein-RNA complexes were excised and then digested with proteinase K. RNAs were purified and cDNAs were made using Superscript III (Invitrogen) with Rclip primers. cDNAs were separated on 6% denaturing urea polyacrylamide gel, then three bands at 120-200 nt (high), 85-120 nt (medium) and 70-85 nt (low) were excised, purified and circularized by Circligase II (Epicenter). Single-stranded cDNAs were hybridized with Cut-Oligo and then digested by BamH I. Linearized cDNAs were PCR amplified and purified. The obtained iCLIP libraries were subjected to high throughput deep sequencing. Linearized cDNAs were PCR amplified and purified on PAGEs to obtain iCLIP libraries, which were subjected to Illumina HiSeq X ten at CAS-MPG Partner Institute for Computational Biology Omics Core, Shanghai, China.