Additional file 1: of Regulation of constitutive and alternative mRNA splicing across the human transcriptome by PRPF8 is determined by 5′ splice site strength

Validation of PRPF8 depletion. a Validation of PRPF8 depletion by qRTPCR. Results are plotted relative to RNA levels in Control siRNA-treated cells, assigned an arbitrary value of 1, and show the mean of triplicate repeats from ten independent experiments ± standard error of the mean. b Exposure to the specific but not control siRNAs efficiently depletes PRPF8 protein in Cal51 cells as detected by western blotting with the indicated antibodies. c, d Characterization of spliceosome iCLIP experiments. UV-crosslinked Cal51 cells were lysed and subjected to partial RNase I digestion (low, final dilution of 1:100,000; high, dilution of 1:5000). Spliceosomal RNPs were immunopurified with anti-SmB/B’ antibody, RNA was 5′ end radiolabeled, and RNPs subjected to denaturing gel electrophoresis and nitrocellulose transfer, an autoradiogram of which is shown. Each lane represents an independent depletion experiment. The interrupted line indicates the area on the nitrocellulose membrane cut out for purification of crosslinked RNP complexes. e Library stats giving the ratio of unique cDNA counts to total cDNA counts for each of the libraries sequenced. The average from three experimental replicates and two technical replicates for each condition is indicated. (PDF 190 kb