In order to purify target RNA molecules to which nElavl proteins are directly bound in vivo we carried out HITS-CLIP with three different anti-nElavl antisera (each of which was specific for the nElavl proteins; see Figure S1A available online). Six independent CLIP experiments using WT and four independent experiments using Elavl3−/− cortical tissue were completed ( Figures 2A–2D). As a negative control, immunoprecipitation was carried out using two different unrelated control antibodies that recognized

cdr2/3 proteins (anti-Yo antisera). We also examined dependence on UV crosslinking by immunoprecipitating nElavl from noncrosslinked tissue. In both of these controls, no signal was detected Alisertib after radio-labeling the immunoprecipitated RNA and analyzing the results by denaturing PAGE ( Figure 2E). Out of 26,190,453 total reads, we obtained 11,966,926 reads that can be unambiguously mapped to unique loci of the reference genome (mm9) (Table S1). Further collapsing of potential PCR duplicates by identical genomic coordinates gave 822,933 unique reads (nElavl

tags) belonging to 81,468 clusters (Tables S1 and S2) (a group of two or more tags overlapping by at least one nt [nucleotide]). In order to determine a set of statistically significant reproducible clusters, for each cluster we calculated a biological complexity coefficient (BC), representing the number of independent experiments that contributed tags to the corresponding cluster, a chi-square score and a false discovery rate (Table S2). To assess Selleck Temozolomide differences in the specificity of three different nElavl antibodies, we determined correlation coefficients (R2) between individual experiments. A high correlation was evident in all pair-wise comparisons of antibodies and in comparison of clusters in WT and Elavl3−/− tissue when we calculated R2 coefficients based on number of tags per 3′UTRs of individual genes (Ab1-Ab1: 0.83 (2 independent experiments), Ab1-Ab2: 0.8, Ab1-Ab3: 0.79, WT-Elavl3−/−: 0.81). In contrast,

comparison of nElavl clusters with those of another neuronally expressed RNA binding protein, Nova ( Licatalosi et al., 2008), resulted in a R2 value of only 0.28, demonstrating the specificity and consistency of CLIP results using individual nElavl Mephenoxalone antibodies. We also calculated R2 values based on number of tags in individual clusters. Since this is a more stringent method of calculation in general we observed lower R2 values ( Table S3). Nonetheless, a higher correlation between the three nElavl antibodies in comparison to nElavl and Nova tags was evident. To gain insight into the potential functional roles nElavl proteins have in RNA regulation, we determined the location of nElavl clusters on target RNA molecules. Analysis of reproducible binding sites with no winnowing of data (all 81,468 clusters) demonstrated that the majority (68.3%) mapped to mRNA-encoding genes, while many (31.