Transcriptome-wide mapping of hnRNP C and U2AF65 using iCLIP
Transcriptome-wide mapping of hnRNP C and U2AF65 using iCLIP
Protein extracts were prepared from UV-crosslinked (UV+) control HeLa cells (Ctrl) and HNRNPC knockdown cells (KD1 and KD2), and RNA was partially digested using low or high concentrations of RNase. RBP-RNA complexes were immuno-purified with a mouse antibody against hnRNP C or U2AF65. To allow visualization of the protein-RNA complexes, the 5' ends of the RNAs were radioactively labeled. The complexes were size-separated using denaturing gel electrophoresis and transferred to a nitrocellulose membrane. RNA recovered by proteinase K digestion from the  nitrocellulose membrane was reverse transcribed and size-selected using denaturing gel electrophoresis. Three of the cDNA size fractions (H: 100-170 nt; M: 85-100 nt; L: 75-85 nt) were further processed and PCR amplified (17 cycles of amplification) to obtain the iCLIP libraries.
Experimental Factor: genetic modification
wild type