Rapid creation and quantitative monitoring of high coverage shRNA libraries

Abstract

Short hairpin RNA libraries are limited by low efficacy of many shRNAs and by off-target effects, which give rise to false negatives and false positives, respectively. Here we present a strategy for rapidly creating expanded shRNA pools (approximately 30 shRNAs per gene) that are analyzed by deep sequencing (EXPAND). This approach enables identification of multiple effective target-specific shRNAs from a complex pool, allowing a rigorous statistical evaluation of true hits.

Figure 1. Schematic for Microarray Synthesis, Cloning, and Enrichment

Microarrays containing 22,000 shRNAs with ∼30 shRNAs per gene are synthesized, and released from the array into a single pool. shRNAs are amplified, cloned, and packaged into lentivirus. Cells infected with the lentiviral shRNA library are sorted by FACS according to phenotype. shRNAs from different fractions are PCR amplified and quantitated either by microarray hybridization or deep sequencing.

Figure 2. Raji cells sorted for low CD45 expression are enriched for anti-CD45 shRNAs

Raji B cells were infected with the CD antigen shRNA library (top panels) or control virus (no insert, lower panels), allowed to grow for 7 days, and sorted by FACS for reduced expression of CD45 and expression of mCherry in two rounds (7 days between sorts). Boxes indicate the gates used to sort CD45-downregulated cells. The percentage of mCherry+ cells displaying reduced CD45 expression is indicated in the figure.

Figure 3. Binned flow-sorting coupled with deep sequencing can quantitatively resolve active shRNAs after a single sort

(a) Raji B cells infected with the CD antigen shRNA library were grown for 7 days and sorted into the indicated fractions. (b) Genomic DNA was prepared from cells collected from the fractions in A, and the shRNAs were PCR amplified and deep sequenced. The percentage of total reads for each shRNA in each fraction for CD45 is depicted; shRNAs are sorted in order of abundance in fraction 1. (c) The abundance of each shRNA in fraction 1 was normalized to its abundance in fraction 5. Only CD45 gave a significant P value (< 2 × 10⁻⁷).