Pharmacological and functional genomic screens play an essential role in the discovery and characterization of therapeutic targets and associated pharmacological inhibitors. Although these screens affect thousands of gene products, the typical readout is based on low complexity rather than genome-wide assays. To address this limitation, researchers from Columbia University Medical Center introduce pooled library amplification for transcriptome expression (PLATE-Seq), a low-cost, genome-wide mRNA profiling methodology specifically designed to complement high-throughput screening assays. Introduction of sample-specific barcodes during reverse transcription supports pooled library construction and low-depth sequencing that is 10- to 20-fold less expensive than conventional RNA-Seq. The use of network-based algorithms to infer protein activity from PLATE-Seq data results in comparable reproducibility to 30 M read sequencing. Indeed, PLATE-Seq reproducibility compares favorably to other large-scale perturbational profiling studies such as the connectivity map and library of integrated network-based cellular signatures.
Schematic illustration of PLATE-Seq workflow
a After conducting a screen in multi-well plates, we lyse the cells and capture mRNA from the cell lysate using an oligo(dT)-coated capture plate. The purified mRNA is then reverse transcribed with barcoded, adapter-linked olig(dT) primers and the resulting cDNA is pooled. All of these steps are automated. The remaining steps, which take place on a single pooled sample, are conducted manually and include cDNA purification, second-strand synthesis, and PCR enrichment. b Molecular-level schematic for constructing 3′-end PLATE-Seq libraries. After reverse transcription with oligo(dT), second-strand synthesis of the pooled cDNA is accomplished using random hexamer primers prior to PCR enrichment of the barcoded pool.