GSM2545764: Flag-NF90 iCLIP rep2; Homo sapiens; RIP-Seq
For iCLIP-seq, The UV-crosslinked cells were resuspended in lysis buffer and sonicated for 20s x 6 times on 25% duty. by immunoprecipitaiton with 8 µg anti-Flag (ShangHai Genomics) antibody at 4°C for 2 hours. Beads with protein-RNA complexes with 5 µg yeast tRNAs were treated with 5 Kunz U/ml of micrococcal nuclease for 5 min at 37°C, and RNA 3' ends were dephosphorylated by PNK at pH6.5. Then pre-adenylated linker L3 (3' modification with biotin) was linked to 3' end of RNA at 16°C overnight. 5' end of RNA was labeled with [γ- 32P] ATP and eluted with 1x Nupage loading buffer (Invitrogen) at 70°C for 10 min. Protein-RNA complexes were eluted with 1xNuPAGE loading buffer. Complexes were separated on 4-12% NuPAGE Bis-Tris gel (Invitrogen), transferred to a nitrocellulose membrane and exposure to a Fuji film at -80℃. Protein-RNA complexes were digested with proteinase K and RNA were reverse transcribed by Superscript III reverse transcriptase (Invitrogen) with Rclip primers. cDNAs were separated on 6% TBE-urea gel, then three bands were cut at 120-200 nt (high), 85-120 nt (medium) and 70-85 nt (low). cDNAs were circularized by Circligase II (Epicentre). Single-stranded cDNAs were hybridized with cut_oligo, and then digested by BamHI. Linearized cDNAs were PCR amplified and purified on PAGEs to obtain iCLIP libraries. For iCLIP-seq, the protein-RNA complexes were treated with 5 Kunz U/ml of micrococcal nuclease with 5 μg yeast tRNAs for 5 min at 37°C, and RNA 3' ends were dephosphorylated by PNK at pH6.5. Then pre-adenylated linker L3 (3' modification with biotin) was linked to 3' end of RNA at 16°C overnight. 5' end of RNA was labeled with [γ-32P] ATP and eluted with 1x Nupage loading buffer (Invitrogen) at 70°C for 10 min. Complexes were separated on 4-12% NuPAGE Bis-Tris gel (Invitrogen), transferred to a nitrocellulose membrane and exposure to a Fuji film at -80℃. Protein-RNA complexes were digested with proteinase K, and RNA were reverse transcribed by Superscript III reverse transcriptase (Invitrogen) with Rclip primers. cDNAs were separated on 6% TBE-urea gel, then three bands were cut at 120-200 nt (high), 85-120 nt (medium) and 70-85 nt (low).cDNAs were circularized by Circligase II (Epicentre). Single-stranded cDNAs were hybridized with cut_oligo, and then digested by BamHI. Linearized cDNAs were PCR amplified and purified on PAGEs to obtain iCLIP libraries, which were subjected to Illumina HiSeq 2500 at CAS-MPG Partner Institute for Computational Biology Omics Core, Shanghai, China.