Rbm15-irCLIP-seq. Rbm15-irCLIP-seq

we performed infrared crosslinking immunoprecipitation followed by sequencing (irCLIP-seq) (Zarnegar et al., 2016) for Rbm15 to directly map its binding sites on RNA. As a principle of concept, the cells were engineered to simultaneously express emGFP-Rbm15 and Xist RNA together, as Rbm15 strongly interacts with Xist A-repeat to deposit the m6A methylation downstream. Cross-linking induced truncation site (CITS or RT stops) is the main signature occurring at our irCLIP-seq datasets. RNA meta-profile plot against normalized transcripts shows that these CITSs reside across the transcript, with 2 main peaks in transcript starts and near the stop-codon regions, in agreement with the RBM15/15B binding profile in human cells (Patil et al., 2016).Motif analysis against CITSs revealed that Rbm15 binding sites prefer U-rich stretches, namely 3 or 4 consecutive Us. This is also true for crosslinking induced mutations (CIMS). Overall design: We performed the ChrMeRIP-seq and identified 6-10% of m6A peaks are located in the intronic regions. We would like to know whether these intronic m6A peaks are also installed by Rbm15-Mettl3/14 complex, we thus performed the Rbm15 irCLIP-seq to examine this pattern.