Extended Data Fig. 2: IP validation from eCLIP experiments, correlation between eCLIP libraries, and de novo sequence motifs and metagene maps for candidate RBPs.
From: Large-scale tethered function assays identify factors that regulate mRNA stability and translation
a,b, In-line western blots of eCLIP IPs of candidate RBPs. Extracts from HEK293T cells (a) or HEK293T cells transfected with the indicated V5-tagged RBP ORFs (b) immunoprecipitated with nonimmune (IgG) control antibodies, and western blot analysis using either RBP-specific (a) or anti-V5 (b) antibodies. The molecular weights (in kilodaltons) of standards are indicated on the right. Arrowheads indicate the calculated molecular weight for each RBP or RBP-V5 fusion protein. c, Heatmap of the Pearson correlation coefficients of fold enrichment of eCLIP peaks for the indicated 14 RBPs analyzed in duplicate. d, De novo sequence motifs in significant eCLIP peaks of the indicated RBP candidates enriched above background, with associated binomial P value. e–h, Metagene maps showing the distribution of eCLIP peak densities at target transcripts. The x axis indicates the relative length of each region. Dark red lines indicate the average number of significantly enriched peaks (≥4-fold enriched and P ≤ 10−2 versus SMInput) of eCLIP peak densities at all transcripts for BOLL, IFIT2, MEX3C, AIMP1 and CNOT7 (e), which show peak enrichment in 3ʹ UTR; DDX6, TOB1, NANOS3 and TOB2 (f), which show peak enrichment in 5ʹ UTR/3ʹ UTR; PARN and CLK3 (g), which show peak enrichment in 5ʹ UTR; and UBAP2L and MTDH (h), which show peak enrichment in CDS. Light shaded areas denote the 95% confidence interval.
