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dc.contributor.authorShrestha, Shaleenen_US
dc.contributor.authorLiu, Xingen_US
dc.contributor.authorSantoso, Clarissaen_US
dc.contributor.authorFuxman Bass, Juanen_US
dc.date2018-11-06
dc.date.accessioned2019-05-14T20:40:24Z
dc.date.available2019-05-14T20:40:24Z
dc.date.issued2019-02-11
dc.identifier.citationShaleen Shrestha, Xing Liu, Clarissa Santoso, Juan Fuxman Bass. 2019. "Enhanced yeast one-hybrid screens to identify transcription factor binding to human DNA sequences." Journal of Visualized Experiments, 144, e59192. https://doi.org/10.3791/59192
dc.identifier.issn1940-087X
dc.identifier.urihttps://hdl.handle.net/2144/35601
dc.description.abstractIdentifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and computational approaches. One such method is the yeast one-hybrid (Y1H) assay, in which interactions between TFs and DNA regions are tested in the milieu of the yeast nucleus using reporter genes. Y1H assays involve two components: a 'DNA-bait' (e.g., promoters, enhancers, silencers, etc.) and a 'TF-prey,' which can be screened for reporter gene activation. Most published protocols for performing Y1H screens are based on transforming TF-prey libraries or arrays into DNA-bait yeast strains. Here, we describe a pipeline, called enhanced Y1H (eY1H) assays, where TF-DNA interactions are interrogated by mating DNA-bait strains with an arrayed collection of TF-prey strains using a high density array (HDA) robotic platform that allows screening in a 1,536 colony format. This allows for a dramatic increase in throughput (60 DNA-bait sequences against >1,000 TFs takes two weeks per researcher) and reproducibility. We illustrate the different types of expected results by testing human promoter sequences against an array of 1,086 human TFs, as well as examples of issues that can arise during screens and how to troubleshoot them.
dc.publisherJournal of Visualized Experimentsen_US
dc.relation.ispartofJournal of Visualized Experimentsen_US
dc.subjectTranscription factors
dc.subjectDNA
dc.subjectHuman
dc.titleEnhanced yeast one-hybrid screens to identify transcription factor binding to human DNA sequencesen_US
dc.typeArticle
dc.description.versionAccepted manuscripten_US
dc.identifier.doi10.3791/59192
dc.identifier.pmid30799854
pubs.elements-sourcemanual-entryen_US
pubs.notesEmbargo: Not knownen_US
pubs.organisational-groupBoston Universityen_US
pubs.organisational-groupBoston University, College of Arts & Sciencesen_US
pubs.organisational-groupBoston University, College of Arts & Sciences, Department of Biologyen_US
pubs.publication-statusPublisheden_US


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