Life Science

Genome-wide Screen for miRNA Targets Using the MISSION® Target ID Library

By: Matthew J. Cousens, Kevin Forbes, Carol Kreader, Jack Sago Carrie Cupp, John Swarthout

The MISSION Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The MISSION Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see workflow).

To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3΄TKzeo dual-selection plasmid (see vector map). An Excel file of the gene targets represented in the library is available for download. Results from Illumina sequencing, show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

Please visit the MISSION Target-ID Library page to learn more.