2C-CLIP_method development and validation

In this experiment we test our recently developed 2C-CLIP method for the identification of the RNA molecules bound by specific RNA binding proteins. 2C-CLIP, involves a first round of 2C, the treatment of the eluates with DNase I and an RNA fragmentation step. After this, a small aliquot is separated to be sequenced as an input, while the rest is subjected for immunoprecipitation of the protein of interest. Libraries from the input and the RNA isolated from the immunoprecipitation are generated and sequenced. In this particular experiment we used Pab1-TAP and Tdh3-Protein A tagged strains to be used in IgG-based pulldowns. As a negative control, an untagged WT strain was also included in the experiment. Sequencing data from the inputs was used to address the variability in gene expression between strains and sequencing data from the IPs to detect enriched target genes or regions.