Dataset Information

Pooled-matrix protein interaction screens using Barcode Fusion Genetics.

ABSTRACT: High-throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome-scale interaction mapping. Here, we report Barcode Fusion Genetics-Yeast Two-Hybrid (BFG-Y2H), by which a full matrix of protein pairs can be screened in a single multiplexed strain pool. BFG-Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein-pair barcodes that can be quantified via next-generation sequencing. We applied BFG-Y2H to four different matrices ranging in scale from ~25 K to 2.5 M protein pairs. The results show that BFG-Y2H increases the efficiency of protein matrix screening, with quality that is on par with state-of-the-art Y2H methods.

SUBMITTER: Yachie N

PROVIDER: S-EPMC4848762 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Publications

Pooled-matrix protein interaction screens using Barcode Fusion Genetics.

Yachie Nozomu N Petsalaki Evangelia E Mellor Joseph C JC Weile Jochen J Jacob Yves Y Verby Marta M Ozturk Sedide B SB Li Siyang S Cote Atina G AG Mosca Roberto R Knapp Jennifer J JJ Ko Minjeong M Yu Analyn A Gebbia Marinella M Sahni Nidhi N Yi Song S Tyagi Tanya T Sheykhkarimli Dayag D Roth Jonathan F JF Wong Cassandra C Musa Louai L Snider Jamie J Liu Yi-Chun YC Yu Haiyuan H Braun Pascal P Stagljar Igor I Hao Tong T Calderwood Michael A MA Pelletier Laurence L Aloy Patrick P Hill David E DE Vidal Marc M Roth Frederick P FP

Molecular systems biology 20160422 4

High-throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome-scale interaction mapping. Here, we report Barcode Fusion Genetics-Yeast Two-Hybrid (BF ...[more]

PMID: 27107012

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Project description:BackgroundRNAi screens via pooled short hairpin RNAs (shRNAs) have recently become a powerful tool for the identification of essential genes in mammalian cells. In the past years, several pooled large-scale shRNA screens have identified a variety of genes involved in cancer cell proliferation. All of those studies employed microarray analysis, utilizing either the shRNA's half hairpin sequence or an additional shRNA-associated 60 nt barcode sequence as a molecular tag. Here we describe a novel method to decode pooled RNAi screens, namely barcode tiling array analysis, and demonstrate how this approach can be used to precisely quantify the abundance of individual shRNAs from a pool.ResultsWe synthesized DNA microarrays with six overlapping 25 nt long tiling probes complementary to each unique 60 nt molecular barcode sequence associated with every shRNA expression construct. By analyzing dilution series of expression constructs we show how our approach allows quantification of shRNA abundance from a pool and how it clearly outperforms the commonly used analysis via the shRNA's half hairpin sequences. We further demonstrate how barcode tiling arrays can be used to predict anti-proliferative effects of individual shRNAs from pooled negative selection screens. Out of a pool of 305 shRNAs, we identified 28 candidate shRNAs to fully or partially impair the viability of the breast carcinoma cell line MDA-MB-231. Individual validation of a subset of eleven shRNA expression constructs with potential inhibitory, as well as non-inhibitory, effects on the cell line proliferation provides further evidence for the accuracy of the barcode tiling approach.ConclusionsIn summary, we present an improved method for the rapid, quantitative and statistically robust analysis of pooled RNAi screens. Our experimental approach, coupled with commercially available lentiviral vector shRNA libraries, has the potential to greatly facilitate the discovery of putative targets for cancer therapy as well as sensitizers of drug toxicity.

Dataset Information

Pooled-matrix protein interaction screens using Barcode Fusion Genetics.

Publications

Pooled-matrix protein interaction screens using Barcode Fusion Genetics.

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