RNA Affinity Purification followed by sequencing (RAP-seq) allows identification of transcriptome-wide RBP binding sites using recombinant RBPs and total purified cellular RNA
RAP-seq is a new method that provides in vitro derived RNA-Interactomes for any given RBP. In RAP-seq a recombinant RBP is produced as fusion with a HaloTag which is used to recover and purify the RBP of interest. The RBP-Halo fusion is then incubated with fragmented total RNA derived from any given sample of interest. The bound RNA fragments are subsequently eluted and cloned using a small RNA library preparation protocol for sequencing the pool of bound molecules on an Illumina NGS platform.   In this study, RAP-seq was used to identify RNA-Interactomes of 26 novel RBPs (aka non-canonical RBPs) newly discovered in proteome-wide studies as RNA binders. RAP-seq was also used to profile vertebrate HuR orthologs and described the biochemical evolutionary differences and similarities of the 6 orthologs profiled. Cancer associated IGF2BP1, IGF2BP2 and IGF2BP3 variants were also profiled and transcriptome-wide changes in their RNA Interactomes with respect to the wild-type IGF2BPs were reported. Also a transcriptome-wide cooperative binding assay was perfomed to evaluate the cooperative roles of HuR and PTBP1 in binding to their native RNA targets.  In addition, a typical RAP-seq substrate (fragmented total RNA) if reverse-transcribed into cDNA and than in vitro transcribed again using a T7 RNA Polymerase can be depleted of any native endogenous RNA modifications and RAP-seq assyas perfomed in parallel with the native substrate and the T7 RNAP produced one allowed us to discern the m6A dependency in transcriptome-wide binding events for YTHDF1, hence with RAP-seq we also report T7-RAP-seq.