Supplementary Figure 1: AGO2-HITS-CLIP.

(a) Top left, SDS-PAGE of RNA-AGO2 or control IPs with nonspecific IgG were transferred to nitrocellulose and exposed to X-Ray film to reveal P32-RNAs bound to AGO2 (dotted square). P32 RNAs were isolated (dotted line) from nitrocellulose membranes and cDNAs generated with specific primers containing an Illumina barcode for high throughput sequencing (Methods). AGO2-mRNA complexes appear as ~200 nt bands (70 nt AGO-mRNA + 120 nt Illumina primer), while AGO2-miRNAs are ~150 nt (30 nt miRNA+120 nt Illumina primer). DNA MW markers are indicated in the left lane of the gel. Bottom left, Integrative Genomics Viewer display of HITS-CLIP reads. Right, computational pipeline to identify reads from miRNAs and mRNAs in AGO2 IPs. Three replicates for each cell type (HUAEC and HUVEC) were processed as described in Fig.1a. 68 million (M) total reads were analyzed. Upon removal of Illumina adapters and duplicates from PCR-based library preparation, reads were mapped to the human genome (UCSC hg19), resulting in 1M unique reads for each cell type. Reads were mapped to miRBase to identify miRNAs and processed using Piranha software to identify significant AGO2-binding sites (peaks) (methods). (b) Left, Bar graph of the Log(FDR) for the significant enriched Gene Ontology terms resulting from the HITS-CLIP assay and identified by DAVID software. Right, Bar graph of the Log(FDR) for the significant enriched Gene Ontology terms resulting from the microarray analysis of endothelial cells in culture versus freshly isolated.