GSM6793308: imb_koenig_2018_01_sub12, in vitro oligo library, 50nM U2AF2, rep1; synthetic construct; RIP-Seq
In vitro iCLIP measures the intrinsic binding affinity of a given RBP.  Recombinant protein and minigene in vitro transcripts (PMID: 29643205) or a large-scale oligo library are mixed and subjected to crosslinking and immunoprecipitation of the RBP of interest. For all in vitro iCLIPs, the in vitro transcript pool or oligo library was preheated for 5 min at 70°C to minimise RNA secondary structures.For minigene in vitro iCLIP, a final concentration of 2 nM of in vitro transcript pool were added to 50 nM U2AF2 RRM12 alone, or to 50 nM U2AF2 RRM12 supplemented with either 50 nM of FUBP1FL, FUBP1ΔN, or FUBP1N74 (imb_koenig_2018_01_sub12). The mixtures were incubated at 37°C for 5 min before UV-irradiation (50 mJ/cm^2). Minigene in vitro iCLIP was spiked with 10 µl crosslinked mixture containing 250 nM U2AF2RRM12 and 6 nM NUP133 in vitro transcript for normalisation.For the oligo in vitro iCLIP, the large-scale oligo library of 200mers was used. Here, 50 nM of the oligo library was incubated with 50 nM U2AF2 RRM12 alone or with either 50 nM or 300 nM FUBP1 (imb_koenig_2018_01_sub16). The mixtures were incubated at 37°C for 5 min before UV-irradiation (50 mJ/cm^2). Partial RNase digestion and L3 linker ligation was skipped as they do not apply here. Oligo in vitro iCLIP was spiked with a mix of ten 150 nt long spike-in oligonucleotides for normalisation. Reverse Transcription and library generation was performed according to the iCLIP2 protocol (PMID: 31610236). In vitro iCLIP libraries were sequenced on an Illumina NextSeq 500 sequencing machine as 150 nt single-end reads including a 6 nt sample barcode as well as 5+4 nt unique molecular identifiers (UMIs).