mixed sample
Protocols: Previously constructed cell lines harboring MRB8170 and MRB4160 with modified TAP-tags, plus parental cell lines (29-13 cells) were collected after 48 hours of induction using tetracycline. All cell lines were grown in SDM-79 medium and were maintained in the exponential mid-log growth phase at 27°C. Protein extracts were prepared from UV-crosslinked (MRB8170, MRB4160 and MRP1) and control (UV crosslinked parental cell), and RNA was partially digested using low concentration of RNAse. RBP-RNA complexes were purified and subsequently visualized by 5' end radiolabelling of RNAs. The complexes were size separated using denaturing gel electrophoresis and transferred to nitrocellulose membrane, Recovered RNA from the nitrocellulose membrane was reverse transcribed and size selected using denaturing gel electrophoresis. The size selected cDNA were further processed and PCR amplified (20 cycles) to obtain the iCLIP libraries. The library preparation was done according to the previously published protocol (Huppertz et al, Methods, 2014)