Homo sapiens
Protocols: MCF7 cells were cultured in 10 cm diameter dishes. When cells reached confluence, media was removed and cells were washed twice with ice-cold PBS 1X. 6ml of ice cold PBS 1X were added, plates were placed on ice and irradiated once at 1500mJ/cm2 254 nm. Cell lysis, immunoprecipitation, 3' end adapter ligation, 5' end radioactive labelling were performed as described in Huppertz, I., et al., iCLIP: protein-RNA interactions at nucleotide resolution. Methods, 2014. protein-bound RNA isolation were performed as described in Huppertz, I., et al., iCLIP: protein-RNA interactions at nucleotide resolution. Methods, 2014. iCLIP library preparation was performed as described in Huppertz, I., et al., iCLIP: protein-RNA interactions at nucleotide resolution. Methods, 2014. The following Retro Transcription oligos (RToligos) were used to insert differential barcodes for each sample: Rt8clip (DDX3X rep1) /5Phos/NNCATTNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC Rt9clip (DDX3X rep2) /5Phos/NNGCCANNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC Rt11clip(DDX54rep1) 5Phos/NNGGTTNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC Rt13clip(DDX54rep2) /5Phos/NNTCCGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC Rt6clip (Rb IgG) /5Phos/NNCCGGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC Rt10clip(MsIgG) /5Phos/NNGACCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC RToligos contain sequences for P5/P7 Illumina adapters for library PCR amplification and following high-throuput sequencing. A sample-specific adapter sequence is located immediately following the P7 adapter sequence. These barcodes are 9-mers of the format NNNXXXXNN, where XXXX was a fixed 4 bp barcode for each sample that allow de-multiplexing, and the Ns were randomly incorporated bases that allow PCR duplicates removal. iCLIP library preparation was performed as described in Huppertz, I., et al., iCLIP: protein-RNA interactions at nucleotide resolution. Methods, 2014.