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Genome Wide RNAi Analysis
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The objective of the Genome-wide RNAi Analysis Core is to advance the research programs of BCM-IDDRC investigators through the use of high-throughput RNA interference (RNAi) technologies. RNAi has emerged as a robust tool for loss-of-function genetics in human and mouse systems. The Genome-wide RNAi Analysis Core was created to harness the potential of this new technology and facilitate BCM-IDDRC and BCM investigators in using RNAi-based approaches. The Core provides intellectual and technologic resources for BCM-IDDRC investigators in the design and implementation of loss-of-function experiments for studying single genes as well as genome-wide approaches. These resources enable investigators to employ genetic approaches that are often cost-and labor-prohibitive for individual laboratories. Specifically, the Core offers the following resources: RNAi vectors:The Core maintains Open Biosystems® collections of short-hairpin RNA (shRNA) vectors (Figure 1A) targeting nearly all of the protein-coding genes in the human and mouse genomes (84,288 shRNAs and 63,360 shRNAs, respectively). Individual lentiviral shRNA vectors are provided to IDDRC investigators as bacterial cultures, purified plasmid DNA, or lentiviral supernatant (format chosen by investigator) at approximately 12% of commercial costs. Custom RNAi libraries: In addition to individual shRNA vectors, BCM-IDDRC investigators have access to genome-wide shRNA libraries. The core also utilizes robotic instrumentation to assemble custom shRNA collections as pools or 96-well arrays. These collections are distributed in bacterial, DNA, or lentiviral formats. This resource enables functional analysis of candidate gene sets determined by IDDRC investigators using other methods (expression array, genomics, proteomics, informatic predictions, etc.). Design and implementation of genetic screens: To utilize these RNAi-based tools in 96-well arrays, the Core offers both high-throughput flow cytometry and automated inverted fluorescence microscopy. These resources enable phenotypic analysis in 96-well based screens (1 shRNA per well). Notably, Core personnel has substantive experience with automated assays for many phenotypes including, but not limited to, cell proliferation, apoptosis, protein abundance and localization, cellular and organelle morphology, etc. This experience enables Core personnel to effectively support BCM-IDDRC investigators in the implementation of assays that they have developed in their own laboratories. To complement 96-well-based approaches, the Core facilitates genetic screens with pooled shRNA collections via shRNA-barcoding technology (Figure 1B, right panels). Each shRNA vector is tagged with a unique 60 base-pair barcode, thus enabling one to track the abundance of individual shRNAs in complex pools by microarray methods. Using this system, one can simultaneously screen large pools of shRNAs (> 20,000 at a time) in a rapid and cost-efficient manner without the limitations of siRNA approaches (cost and transient phenotypes). Data Management: All data from genetic screens performed in the Core are maintained in a tiered data storage consisting of a high-speed Storage Area Network (SAN). This SAN includes 72 terabytes of primary storage and is backed by a 35 terabyte network that reconstructs data upon primary drive failure. |
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