Additional file 1 of Comprehensive mapping of exon junction complex binding sites reveals universal EJC deposition in Drosophila

Additional file 1. Figure S1. Complexity for human and Drosophila eIF4A3 eCLIP libraries a) Bar plot displaying the read counts at successive steps of read analysis for each sample, including raw reads, uniquely mapped reads, deduplicated reads, and reads that intersect coding exons in the human data. Note that SMInput 2 appears as having 0 reads, due to very low output and plot scale b) The same as a) for Drosophila eCLIP data. c) Venn diagram depicting the similarity of eCLIP binding sites after PureCLIP binding site discovery for all Drosophila replicates. The Jaccard index (JI) on the right of each Venn diagram is calculated based on the values presented in the Venn diagram. Figure S2. Comparison of human and Drosophila transcriptomes and validation of CRISPR editing of Drosophila S2R+ cells a) Distribution of the number of exons per transcript in D. melanogaster and H. sapiens. b) Total number of annotated exons in D. melanogaster and H. sapiens. Drosophila annotations were obtained from the FlyBase consortium, version BDGP6. Human annotations were obtained from Ensembl, version GRCh38; only the longest isoform and with experimental evidence were used for quantification. c) Western blot to validate expression of endogenous eIF4A3 cells fused to 3xHA in S2R+. Anti-HA membrane (right) with lane 1 from a modified and single isolated clone, and lane 2 with wild-type cells. Anti-eIF4A3 Drosophila membrane (left) with lane 1 from a modified and single isolated clone and lane 2 with wild-type cells. The red arrow points to the expected size for endogenous eIF4A3, which becomes fainter in the S2 HA (lane 1). The blue arrow points to the expected size for eIF4A3-HA, which appears only upon CRISPR-cas9 and is absent in the wild type, with expression levels similar to eIF4A3-WT in WT cells. d) Co-IP of Y14-FLAG pulled down by eIF4A3-HA. Lane 1: input, with both proteins expressed. Lane 2: IP of eIF4A3 co-precipitates Y14-FLAG. Figure S3. eCLIP biases a) The scatter plot displays the Stop Rate scores per position for replicates 1 and 2 of Drosophila eIF4A3 eCLIP. The Spearman correlation score is shown on top. b) As in a) but for Drosophila eIF4A3 eCLIP datasets analyzed by PureCLIP. c) Boxplot showing the correlation between expression level and U count for EJC detection. Exons were divided in 10 different classes of equal bins and further divided into 7 classes depending on its U count in the EJC interacting window. SRD scores (y-axis) were plotted. Figure S4. ipaRt biasesa) IGV screenshot comparing eCLIP replicate 1 and ipaRt replicate 1, illustrating differences in both overall enrichment and background noise for the Galk gene. b) Stacked bar plot showing the count of detected exons versus total exons per exon group, including first exons, internal exons and last exons. c) Box plot comparing T counts in the window for detected and undetected exons in ipaRTs.