Abstract
RNA interference (RNAi) screening is a state-of-the-art technology that enables the dissection of biological processes and disease-related phenotypes. The commercial availability of genome-wide, short hairpin RNA (shRNA) libraries has fueled interest in this area but the generation and analysis of these complex data remain a challenge. Here, we describe complete experimental protocols and novel open source computational methodologies, shALIGN and shRNAseq, that allow RNAi screens to be rapidly deconvoluted using next generation sequencing. Our computational pipeline offers efficient screen analysis and the flexibility and scalability to quickly incorporate future developments in shRNA library technology.
Highlights
RNA interference (RNAi) facilitates the assessment of gene function by silencing gene expression using synthetic anti-sense oligonucleotides or plasmids
Physiological RNAi is primarily mediated by non-protein-coding RNA transcripts, known as microRNAs. miRNAs are produced in a similar manner to mRNAs, but miRNAs are processed into shorter RNA species containing a hairpin structure, known as short-hairpin RNAs. short hairpin RNA (shRNA) are in turn processed into short double-stranded pieces of RNA known as short interfering RNAs
We describe how methods were selected from principles and procedures established by McManus [3], Hannon, Elledge and Lowe [2,4,5] and optimized for the entire shRNA barcode screening workflow from library production to statistical analysis (Figure 1)
Summary
RNA interference (RNAi) facilitates the assessment of gene function by silencing gene expression using synthetic anti-sense oligonucleotides or plasmids. It exploits a physiological mechanism that represses gene expression, primarily by causing the degradation of mRNA transcripts. Physiological RNAi is primarily mediated by non-protein-coding RNA transcripts, known as microRNAs (miRNAs). Within the multi-protein RNA-induced silencing complex (RISC), one strand of a siRNA duplex binds a protein-coding mRNA transcript that bears a complementary nucleotide sequence. This interaction allows a nuclease in the RISC to cleave and destroy the proteincoding mRNA, silencing the expression of the gene in a relatively sequence-specific manner. While siRNAs are typically used in multiwell plate-based screening, shRNAs are commonly used for pooled competitive screening approaches, often called barcode screening
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