Abstract
Pooled short-hairpin RNA (shRNA) library screening is a powerful tool for identifying a set of genes in biological pathways that require stable expression to produce a desired phenotype. Massive parallel sequencing of half-hairpins has proven highly variable and has not given satisfactory results concerning the relative abundance of different shRNAs before and after selection. Here, the authors describe a method for quantitative comparison of half-hairpins from pooled shRNAs in the mir30-based pGIPZ vector that is analyzed by massive parallel sequencing. Introducing a multiplexing code and refining the sample preparation scheme resulted in the predicted ability to detect twofold enrichments. These improvements should permit half-hairpin sequencing to analyze either dropout screens or selective pooled shRNA screens of limited stringency to analyze phenotypes not accessible in transient experiments.
Original language | English (US) |
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Pages (from-to) | 258-265 |
Number of pages | 8 |
Journal | Journal of Biomolecular Screening |
Volume | 17 |
Issue number | 2 |
DOIs | |
State | Published - Feb 2012 |
Keywords
- RNA interference
- emulsion PCR
- half-hairpins
- in vivo screening
- multiplex assays
- parallel sequencing
- shRNA libraries
ASJC Scopus subject areas
- Analytical Chemistry
- Biotechnology
- Biochemistry
- Molecular Medicine
- Pharmacology
- Drug Discovery