title: GSM3045720: XPO5_HITS-CLIP_4; Homo sapiens; RIP-Seq
library_construction_protocol: HEK293T cells were UV-crosslinked, re-suspended in lysis buffer.RNA-XPO5 complexes were isolated with Anti-Exportin-5 antibody (Abcam). For XPO5 HITS-CLIP, HEK293FT cell lysates were incubated with Anti-Exportin-5 antibody (Abcam) or control IgG for 1 hr on ice with or without RNase.  After incubation, dynabeads were added into the protein-RNA complexes solutions and incubate for 1h at 4°C, followed by labelling 3′ end of RNA with [γ- 32P] ATP using PNK. Then 5' Ligation Adapter-NN was linked to 5′ end of RNA using T4 RNA ligase 1 at room temperature for 2h. Protein-RNA complexes were incubated in 1x Nu-PAGE loading buffer (Invitrogen) at 70°C for 10 min, resolved on 8% Nu-PAGE Bis-Tris gel (Invitrogen), transferred to a nitrocellulose membrane and exposed to a Fuji film. Bands corresponding to specific protein-RNA complexes were excised and then digested with proteinase K. RNAs were purified and adenylated linker was linked to 3' end of purified RNAs. cDNAs were made using Superscript III(Invitrogen) with 3' adenylated linker primers. cDNAs were separated on 15% denaturing urea polyacrylamide gel and one area around 100 bp were excised purified, followed by amplification by Phusion High Fidelity polymerase (NEB). PCR products were seperated by 6% denaturing urea polyacrylamide gel and three bands around 100-150 were purified. Obtained CLIP libraries were subjected to high throughput deep sequencing.