GSM6594618: iCLIP_PRPF8_HeLa_WT_TTGT_Rep2; Homo sapiens; OTHER
Cells were lysed and treated with RNase I. GFP-tagged proteins were immunoprecipitated with Gamma binding beads coupled to goat anti-EGFP antibody. After stringent washing, RNA was dephosphorylated and ligated to a 3' linker. Protein-RNA complexes were resolved by denaturing gel electrophoresis and transferred to nitrocellulose membrane. A membrane region above the expected size of the protein was cut and RNA was released by Proteinase K treatment. RNA was reverse transcribed using reverse primer containing a barcode sequence. cDNA was size selected by denaturing gel electrophoresis and circularized by ssDNA circligase. A specific oligonucleotide was annealed to create a restriction site for BamHI and cDNA was linearized by digest with BamHI. Linearized cDNA was PCR amplified with Solexa sequencing primers to create the final libraries. For more details see: Huppertz et al., (2014): iCLIP: protein-RNA interactions at nucleotide resolution. Methods. PMID:24184352.