We thank the staff within the Institute of Human Genetics for NGS library preparation and sequencing. We thank Einat Snir for her expertise in preparing genomic library fragments for the Y2H plasmid library made here. This work was supported by National Institutes of Health: NIH R21 EB021870-01A1 and by NSF Research Project Grant: 1517110.

1. Preparation of Media and Plates
NOTE: All plates need to be made minimally 2 days before beginning the protocol. The media can be made at any point. However, the buffered yeast extract peptone dextrose adenine (bYPDA) needs to be made the day of which it will be used. Some media is made using a supplement mix containing a level of adenine that is larger than what is typically used. Most minimal media supplements specify 10 mg/L adenine. Supplements labeled &#39;+40Ade&#39; specify a total of ...

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	<li>Fields, S., Song, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+genetic+system+to+detect+protein-protein+interactions.">A novel genetic system to detect protein-protein interactions.</a> Nature. 340 (6230), 245-246 (1989).</li><li>Bruckner, A., Polge, C., Lentze, N., Auerbach, D., Schlattner, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+two-hybrid,+a+powerful+tool+for+systems+biology.">Yeast two-hybrid, a powerful tool for systems biology.</a> International Journal of Molecular Sciences. 10 (6), 2763-2788 (2009).</li><li>Vidal, M., Fields, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+two-hybrid+assay:+still+finding+connections+after+25+years.">The yeast two-hybrid assay: still finding connections after 25 years.</a> Nature Methods. 11 (12), 1203-1206 (2014).</li><li>Pashkova, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DEEPN+as+an+Approach+for+Batch+Processing+of+Yeast+2-Hybrid+Interactions.">DEEPN as an Approach for Batch Processing of Yeast 2-Hybrid Interactions.</a> Cell Reports. 17 (1), 303-315 (2016).</li><li>Rajagopala, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+Protein-Protein+Interactome+Networks+Using+Yeast+Two-Hybrid+Screens.">Mapping the Protein-Protein Interactome Networks Using Yeast Two-Hybrid Screens.</a> Advances in Experimental Medicine and Biology. 883, 187-214 (2015).</li><li>Weimann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Y2H-seq+approach+defines+the+human+protein+methyltransferase+interactome.">A Y2H-seq approach defines the human protein methyltransferase interactome.</a> Nature Methods. 10 (4), 339-342 (2013).</li><li>Yachie, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pooled-matrix+protein+interaction+screens+using+Barcode+Fusion+Genetics.">Pooled-matrix protein interaction screens using Barcode Fusion Genetics.</a> Molecular Systems Biology. 12 (4), 863 (2016).</li><li>Trigg, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CrY2H-seq:+a+massively+multiplexed+assay+for+deep-coverage+interactome+mapping.">CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping.</a> Nature Methods. , (2017).</li><li>Estojak, J., Brent, R., Golemis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+two-hybrid+affinity+data+with+in+vitro+measurements.">Correlation of two-hybrid affinity data with in vitro measurements.</a> Molecular and Cellular Biology. 15 (10), 5820-5829 (1995).</li><li>Rajagopala, S. V., Hughes, K. T., Uetz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benchmarking+yeast+two-hybrid+systems+using+the+interactions+of+bacterial+motility+proteins.">Benchmarking yeast two-hybrid systems using the interactions of bacterial motility proteins.</a> Proteomics. 9 (23), 5296-5302 (2009).</li><li>James, P., Halladay, J., Craig, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+libraries+and+a+host+strain+designed+for+highly+efficient+two-hybrid+selection+in+yeast.">Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.</a> Genetics. 144 (4), 1425-1436 (1996).</li><li>Barbas, C. F., Burton, D. R., Scott, J. K., Silverman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+DNA+and+RNA.">Quantitation of DNA and RNA.</a> CSH Protoc. 2007, pdb ip47 (2007).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SDS-Polyacrylamide+Gel+Electrophoresis+of+Proteins.">SDS-Polyacrylamide Gel Electrophoresis of Proteins.</a> Cold Spring Harbor Protocols. 2006 (4), (2006).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proceedings of the National Academy of Sciences USA. 76 (9), 4350-4354 (1979).</li><li>Alegria-Schaffer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting+using+chemiluminescent+substrates.">Western blotting using chemiluminescent substrates.</a> Methods in Enzymology. 541, 251-259 (2014).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> Journal of Visualized Experiments. (62), (2012).</li><li>Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+p21+Cdk-interacting+protein+Cip1+is+a+potent+inhibitor+of+G1+cyclin-dependent+kinases.">The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases.</a> Cell. 75 (4), 805-816 (1993).</li></ol>

Here we provide a guide for how to perform Y2H assays in batch using optimized methods. There are a few critical steps in the procedure to help ensure that the population of yeast that would be placed under selection is representative of the starting library and that enough of the starting yeast population is used to undergo selection to limit variability. Importantly, these benchmarks are relatively easy to achieve alongside adapting the methods and materials for a traditional Y2H assay, thus making this approach access...

A complete understanding of cell biological processes relies on finding the protein interaction networks that underlie their molecular mechanisms. One approach to identify protein interactions is the yeast 2-hybrid (Y2H) assay, which works by assembling a functioning chimeric transcription factor once two protein domains of interest bind to one another1. A typical Y2H screen is performed by creating a population of yeast that houses both a library of plasmids encoding interacting proteins fused to a transcriptional activator (e.g., &#39;prey&#39; fusion protein) and a given &#39;bait&#39; plasmid comprised of the protein of interest fused to a DNA binding domain (e.g., the Gal4 DNA-binding domain that binds to the Gal4-upstream activating sequence). One of the main advantages of the Y2H approach is that it is relatively easy and inexpensive to conduct in a typical laboratory equipped for routine molecular biological work2. However, when traditionally performed, a user samples individual colonies that arise upon selection for a positive Y2H interaction. This severely limits the number of library &#39;prey&#39; clones that can be surveyed. This problem is compounded when the abundance of a particular interacting prey is very high relative to the others, diminishing the chance of detecting interaction from low abundance prey plasmids.
One solution for using the Y2H principle in comprehensive coverage of the proteome is the use of a matrix-formatted approach wherein an array containing known individual prey plasmids can be digitally interrogated. However, such an approach requires an infrastructure that is not readily accessible or cost-effective to individual investigators who are interested in defining the interactome of a small number of proteins or domains3. In addition, very complex prey libraries that may encode multiple fragments of interacting proteins would expand the size of such matrix arrays to impractical sizes. An alternative is to perform assays with complex libraries in batches and assess the presence of interacting clones using massive parallel high-throughput sequencing4. This can be applied to assay the presence of prey plasmids that arise in multiple colonies using a typical Y2H formatted approach in which yeast cells housing an interacting pair of fusion proteins are allowed to grow on a plate5,6. This general idea can be accentuated to increase query of both multiple bait and prey components at the same time7,8.
Still, many investigations require an easier yet more focused effort on just a few protein &#39;baits&#39; and can benefit more by an exhaustive and semi-quantitative query of a single complex prey library. We have developed and validated an approach to perform wide-scale protein interaction studies using a Y2H principle in batch format4. This uses the rate of expansion of a particular prey plasmid as a proxy for the relative strength of Y2H interaction9. Deep sequencing of all plasmids within a population subjected to normal growth or selective growth conditions produces a complete map of clones that yield strong and weak Y2H interactions. The repertoire of interactors can be obtained and directly compared across multiple bait plasmids. The resulting workflow termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks) can thus be used to identify differential interactomes from the same prey libraries to identify proteins, allowing comparison between one protein vs. another.
Here, we demonstrate DEEPN and introduce improvements in the laboratory methods that facilitate its use, which are outlined in Figure 1. Significant improvements include:
Generation of prey yeast populations. One of the key requirements of DEEPN is generating populations of yeast with different bait plasmids that have the same distribution of the plasmid prey libraries. Equivalent baseline populations of the prey plasmid library are essential for making accurate comparisons between the interactomes of different baits. This is best achieved when a library plasmid is already housed in a haploid yeast population and moving a given bait plasmid into that population is achieved by mating to produce a diploid. Here, we provide a clear guide in how to make such populations using commercial libraries housed in haploid yeast. Although we found methods that generate a high number of diploids, the overall mating efficiency of these commercial library-containing yeast strains was low. Therefore, we constructed a new strain that can house prey libraries that yields far more diploids per mating reaction.
New set of bait plasmids. Many current plasmids that express &#39;bait&#39; fusion proteins comprised of the protein of interest and a DNA-binding domain are 2&#181;-based, allowing them to amplify their copy number. This copy number can be quite variable in the population and lead to variability in the Y2H transcriptional response. This in turn could skew the ability to gauge the strength of a given protein interaction based on the growth response of cells under selection. This can be partly address by using a low copy plasmid, some of which have been previously described such as the commercially available pDEST3210. We constructed a new bait plasmid (pTEF-GBD) that produces Gal4-DNA-binding domain fusion proteins within a TRP1 centromere-based low copy plasmid carrying the Kanr resistance gene that also allows cloning of bait fragments both upstream and downstream of the Gal4 DNA-binding domain.
New High-Density Y2H fragment library. We constructed a new plasmid to house Y2H prey libraries and used it to build a highly complex Y2H library made of randomly sheared fragments of genomic DNA from Saccharomyces cerevisiae. Sequence analysis showed that this library had over 1 million different elements, far more complex than previously described yeast genomic Y2H plasmid libraries11. With this new library, we were able to show that the DEEPN workflow is robust enough to accommodate complex libraries with many different plasmids in a manner that is reliable and reproducible.

<table>
	<tbody>
		<tr>
			<td>Illumina HiSeq 4000</td>
			<td>Illumina</td>
			<td></td>
			<td>deep sequencing platform</td>
		</tr>
		<tr>
			<td>Monoclonal anti-HA antibodies</td>
			<td>Biolegend</td>
			<td>901514</td>
			<td>Primary Antibody to detect expression of HA in pGal4AD constructs</td>
		</tr>
		<tr>
			<td>Polyclonal anti-myc antibodies</td>
			<td>QED Biosciences Inc</td>
			<td>18826</td>
			<td>Primary Antibody to detect expression of MYC in pTEF-GBD constructs</td>
		</tr>
		<tr>
			<td>NarI</td>
			<td>New England BioLabs</td>
			<td>R0191S</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI-HF</td>
			<td>New England BioLabs</td>
			<td>R3101S</td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI-HF</td>
			<td>New England BioLabs</td>
			<td>R3236S</td>
			<td></td>
		</tr>
		<tr>
			<td>XhoI</td>
			<td>New England BioLabs</td>
			<td>R0146S</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene Glycol 3350, powder</td>
			<td>J.T. Baker</td>
			<td>U2211-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon Sperm DNA</td>
			<td>Trevigen, Inc sold by Fisher Scientific</td>
			<td>50-948-286</td>
			<td>carrier DNA for yeast transformation section 3.2.1.</td>
		</tr>
		<tr>
			<td>Kanamycin Monosulfate</td>
			<td>Research Products International</td>
			<td>K22000</td>
			<td></td>
		</tr>
		<tr>
			<td>LE Agarose</td>
			<td>GeneMate</td>
			<td>E-3120-500</td>
			<td>used for making DNA agarose gels</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Research Products International</td>
			<td>S23025</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Research Products International</td>
			<td>T60060</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Sorbitol</td>
			<td>Research Products International</td>
			<td>S23080</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium Acetate Dihydrate</td>
			<td>MP Biomedicals</td>
			<td>155256</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>ThermoFisher</td>
			<td>C79</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA Sodium Salt</td>
			<td>Research Products International</td>
			<td>E57020</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract Powder</td>
			<td>Research Products International</td>
			<td>Y20020</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Nitrogen Base (ammonium sulfate) w/o amino acids</td>
			<td>Research Products International</td>
			<td>Y20040</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-Trp-Leu+40ADE</td>
			<td>Formedium</td>
			<td>DCS0789</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-Trp-Leu-His+40ADE</td>
			<td>Formedium</td>
			<td>DCS1169</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-Leu-Met</td>
			<td>Formedium</td>
			<td>DCS0549</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-Trp-Met</td>
			<td>Bio 101, Inc</td>
			<td>4520-922</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Methionine</td>
			<td>Formedium</td>
			<td>DOC0168</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Research Products International</td>
			<td>A11500</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Research Products International</td>
			<td>G32045</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td>used for making media plates in section 1</td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Research Products International</td>
			<td>P20240</td>
			<td></td>
		</tr>
		<tr>
			<td>3-amino-1,2,4 Triazole</td>
			<td>Sigma</td>
			<td>A8056</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoehanol (BME)</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymolyase 100T</td>
			<td>USBiological</td>
			<td>Z1004</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sigma</td>
			<td>P8281</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:IAA</td>
			<td>Ambion</td>
			<td>AM9732</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>238074</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Laboratories, INC</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse A</td>
			<td>ThermoFisher</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Research Products International</td>
			<td>U20200</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Research Products International</td>
			<td>L22010</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Gibco</td>
			<td>15506-017</td>
			<td></td>
		</tr>
		<tr>
			<td>bromophenol blue</td>
			<td>Amresco</td>
			<td>449</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibson Assembly Cloning Kit</td>
			<td>New England Biolabs</td>
			<td>E5510S</td>
			<td>Rapid assembly method for cloning of plasmids in section 2</td>
		</tr>
		<tr>
			<td>NEBNext High-Fidelity 2x PCR Master Mix</td>
			<td>New England Biolabs</td>
			<td>M0541S</td>
			<td>Used for amplification of products for Gibson Assembly in Section 2.3 as well assample preparation for DEEPN deep sequencing in section 6.2.1</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Amresco</td>
			<td>0492-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>Used for purification of pcr products in section 6.2.3</td>
		</tr>
		<tr>
			<td>Qiaquick DNA Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td>Used for purification of digested pTEF-GBD in section 2.1</td>
		</tr>
		<tr>
			<td>KAPA Hyper Prep kit</td>
			<td>KAPA Biosystems</td>
			<td>KK8500</td>
			<td>preparation kit for deep sequencing</td>
		</tr>
		<tr>
			<td>Codon optimization</td>
			<td>http://www.jcat.de</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Codon optimization</td>
			<td>https://www.idtdna.com/CodonOpt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>gBlocks</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>DNA fragments used for cloning in Section 2.2</td>
		</tr>
		<tr>
			<td>Strings</td>
			<td>Thermofisher</td>
			<td></td>
			<td>DNA fragments used for cloning in Section 2.2</td>
		</tr>
		<tr>
			<td>GenCatch Plasmid DNA mini-prep Kit</td>
			<td>EPOCH Life Sciences</td>
			<td></td>
			<td>Used to prepare quantities of DNA in Section 2.3</td>
		</tr>
		<tr>
			<td>Covaris E220</td>
			<td>Covaris</td>
			<td></td>
			<td>high performance ultra-sonicator in section 7</td>
		</tr>
		<tr>
			<td>oligo nucelotide 5&#8217;- CGGTCTT 
			CAATTTCTCAAGTTTCAG -3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>used for pcr amplification and sequencing 5&#39; insert pTEF-GBD during plasmid construction</td>
		</tr>
		<tr>
			<td>oligo nucelotide 5&#8217;-GAGTAACG 
			ACATTCCCAGTTGTTC-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>used for pcr amplification and sequencing 5&#39; insert pTEF-GBD during plasmid construction</td>
		</tr>
		<tr>
			<td>oligo nucelotide 5&#8217;-CACCGTAT 
			TTCTGCCACCTCTTCC-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>used for pcr amplification and sequencing 3&#39; insert pTEF-GBD during plasmid construction</td>
		</tr>
		<tr>
			<td>oligo nucelotide 5&#8217;-GCAACCGC 
			ACTATTTGGAGCGCTG-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>used for pcr amplification and sequencing 3&#39; insert pTEF-GBD during plasmid construction</td>
		</tr>
		<tr>
			<td>oligonucleotide 5&#8217;-GTTCCGATG 
			CCTCTGCGAGTG-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>5&#39; Pimer used for insert amplification of pGAL4AD</td>
		</tr>
		<tr>
			<td>oligonucelotide 5&#8217;-GCACATGCT 
			AGCGTCAAATACC-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>3&#39; Pimer used for insert amplification of pGAL4AD</td>
		</tr>
		<tr>
			<td>oligonucelotide 5&#8217;-ACCCAAGCA 
			GTGGTATCAACG-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>5&#39; Pimer used for insert amplification of pGADT7</td>
		</tr>
		<tr>
			<td>oligonucelotide 5&#8217;- TATTTAGA 
			AGTGTCAACAACGTA -3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>3&#39; Pimer used for insert amplification of pGADT7</td>
		</tr>
		<tr>
			<td>PJ69-4A MatA yeast strain</td>
			<td>http://depts.washington.edu/yeastrc/ James P, Halladay J, Craig EA: Genomic Libraries and a host strain designed for highly efficient two-hybrid selection in yeast. GENETICS 1996 144:1425-1436</td>
			<td></td>
			<td>MATA leu2-3,112 ura3-52 trp1-901 his3-200 gal4D, gal80D, GAL-ADE2 lys2::GAL1-HIS3 met2::GAL7</td>
		</tr>
		<tr>
			<td>pTEF-GBD</td>
			<td>Dr. Robert Piper Lab</td>
			<td></td>
			<td>Gal4-DNA binding doimain expression plasmid</td>
		</tr>
		<tr>
			<td>PLY5725 MATalpha yeast strain</td>
			<td>Dr. Robert Piper Lab</td>
			<td></td>
			<td>MATalpha his3&#8710;1 leu2&#8710;0 lys2&#8710;0 ura3&#8710;0 gal4&#8710; trp1&#8710; Gal80&#8710;</td>
		</tr>
		<tr>
			<td>pGal4AD (pPL6343)</td>
			<td>Dr. Robert Piper Lab</td>
			<td></td>
			<td>Gal4-activation domain expression plasmid</td>
		</tr>
		<tr>
			<td>100 mm petri dishes</td>
			<td>Kord-Vallmark sold by VWR</td>
			<td>2900</td>
			<td></td>
		</tr>
		<tr>
			<td>125 mL PYREX Erlenmeyer flask</td>
			<td>Fisher Scientific</td>
			<td>S63270</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL PYREX Erlenmeyer flask</td>
			<td>Fisher Scientific</td>
			<td>S63271</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 mL PYREX Erlenmeyer flask</td>
			<td>Fisher Scientific</td>
			<td>S63274</td>
			<td></td>
		</tr>
		<tr>
			<td>2,000 mL PYREX Erlenmeyer flask</td>
			<td>Fisher Scientific</td>
			<td>S63275</td>
			<td></td>
		</tr>
		<tr>
			<td>20 X 150 mm Disposable Culture Tube</td>
			<td>Thermofisher</td>
			<td>14-961-33</td>
			<td></td>
		</tr>
		<tr>
			<td>pipet-aid</td>
			<td>Drummond</td>
			<td>4-000-100</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Serological Pipette</td>
			<td>Denville</td>
			<td>P7127</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Serological Pipette</td>
			<td>Denville</td>
			<td>P7128</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL Serological Pipette</td>
			<td>Denville</td>
			<td>P7129</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 mL PYREX Griffin Beaker</td>
			<td>Fisher Scientific</td>
			<td>02-540P</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 mL PYREX Reuasable Media Storage Bottle</td>
			<td>Fisher Scientific</td>
			<td>06-414-1D</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 mL graduated cylinder</td>
			<td>Fisher Scientific</td>
			<td>08-572-6G</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax 190</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>used to measure the Optical Density of cells</td>
		</tr>
		<tr>
			<td>NanoDrop 2000</td>
			<td>Thermo Scientific</td>
			<td>ND-2000</td>
			<td>Spectrophotometer used to quantify amount of DNA</td>
		</tr>
		<tr>
			<td>Electronic UV transilluminator</td>
			<td>Ultra Lum</td>
			<td>MEB 20</td>
			<td>used to visualize DNA in an Ethidium Bromide agarose gel</td>
		</tr>
		<tr>
			<td>P1000 Gilson PIPETMAN</td>
			<td>Fisher Scientific</td>
			<td>F123602G</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 Gilson PIPETMAN</td>
			<td>Fisher Scientific</td>
			<td>F123601G</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 Gilson PIPETMAN</td>
			<td>Fisher Scientific</td>
			<td>F123600G</td>
			<td></td>
		</tr>
		<tr>
			<td>P10 Gilson PIPETMAN</td>
			<td>Fisher Scientific</td>
			<td>F144802G</td>
			<td></td>
		</tr>
		<tr>
			<td>1250 &#181;L Low Retention Pipette Tips</td>
			<td>GeneMate</td>
			<td>P-1236-1250</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;LLow Retention Pipette Tips</td>
			<td>VWR</td>
			<td>10017-044</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L XL Low Retention Pipette Tips</td>
			<td>VWR</td>
			<td>10017-042</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>VWR</td>
			<td>490001-627</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>VWR</td>
			<td>490001-621</td>
			<td></td>
		</tr>
		<tr>
			<td>cell scraper</td>
			<td>Denville Scientific</td>
			<td>TC9310</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fluka Analytical</td>
			<td>318949-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>J.T. Baker</td>
			<td>5674-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Wooden applicators</td>
			<td>Solon Care</td>
			<td>55900</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf microcentrifuge 5424</td>
			<td>Fisher Scientific</td>
			<td>05-400-005</td>
			<td>microcentrifuge</td>
		</tr>
		<tr>
			<td>Sorvall ST16R</td>
			<td>Thermo Fisher Scientific</td>
			<td>75004381</td>
			<td>benchtop centrifuge</td>
		</tr>
		<tr>
			<td>Amersham ECL Rabbit IgG, HRP-linked whole Ab (from donkey)</td>
			<td>GE Healthcare</td>
			<td>NA934-1ML</td>
			<td>Secondary Antibody</td>
		</tr>
		<tr>
			<td>Amersham ECL Mouse IgG, HRP-linked whole Ab (from sheep)</td>
			<td>GE Healthcare</td>
			<td>NA931-1ML</td>
			<td>Secondary Antibody</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>34080</td>
			<td>ECL detection solution</td>
		</tr>
		<tr>
			<td>Isotemp Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Incubator</td>
		</tr>
		<tr>
			<td>Mutitron 2</td>
			<td>INFORS HT</td>
			<td></td>
			<td>Shaking incubator</td>
		</tr>
		<tr>
			<td>Isotemp Digital-Control Water Bath Model 205</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>water bath</td>
		</tr>
		<tr>
			<td>Y2H mouse cDNA library in Y187 (pan tissue)</td>
			<td>Clontech</td>
			<td>630482</td>
			<td>commercially available cDNA Library</td>
		</tr>
		<tr>
			<td>Y2H mouse cDNA library in Y187 (mouse brain)</td>
			<td>Clontech</td>
			<td>630488</td>
			<td>commercially available cDNA Library</td>
		</tr>
		<tr>
			<td>pGADT7 AD Vector</td>
			<td>Clontech</td>
			<td>630442</td>
			<td>commercially available AD Vector housing many cDNA libraries</td>
		</tr>
		<tr>
			<td>pGBKT7 DNA-BD Vector</td>
			<td>Clontech</td>
			<td>630443</td>
			<td>commercially available DNA-BD Vector</td>
		</tr>
		<tr>
			<td>Biolase DNA Polymerase</td>
			<td>Bioline</td>
			<td>BIO-21042</td>
			<td>DNA polymerase used for section 2.4</td>
		</tr>
		<tr>
			<td>GeneMate GCL-60 Thermal Cycler</td>
			<td>BioExpress</td>
			<td>P-6050-60</td>
			<td>pcr machine</td>
		</tr>
		<tr>
			<td>TempAssure 0.5 mL PCR tubes</td>
			<td>USA Scientific</td>
			<td>1405-8100</td>
			<td></td>
		</tr>
	</tbody>
</table>

a yeast 2-hybrid screen in batch to compare protein interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

The Y2H assay has been widely used for finding protein:protein interactions and several adaptations and systems have been developed. For the most part, the same considerations that help ensure success with these previous approaches are important for DEEPN. Some of the important benchmarks include: ensuring expression of DNA-binding domain fusion proteins, ensuring a low background of spurious His+ growth in the diploids containing the bait of interest with an empty prey plasmid, a high ma...

Watch this Scientific Journal Video about A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions at JoVE.com

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

This method uses the principle of the yeast 2-hybrid system in both populations to identify protein-protein interactions that answer key questions about a variety of molecular and cellular processes. This technique leverages deep sequencing to see the interactions of one protein versus another. Since this approach is geared to make parallel comparisons, it can identify sets of proteins that interact with one protein versus another or different confirmations of the same protein or while typing a disease causing mutant version, to reveal differential interactomes.A new yeast 2-hybrid bait plasmid pTEF-GBD was constructed for this procedure as described in the text protocol. This plasmid produces Gal4 DNA binding domain fusion proteins within a TRP1 centromere based low copy plasmid carrying the kanamycin resistance gene that also allows cloning of bait fragments both upstream and downstream of the Gal4 DNA binding domain. This bait plasmid was used to transform MATa yeast, and expression of the Gal4 bait fusion proteins was verified by immunoblotting with anti-MIC antibodies.In addition, a new yeast 2-hybrid library in the streamlined prey plasmid vector PGAL4 AD was created. Genomic DNA was fragmented by shearing, size selected, modified with adapters, and inserted into PGAL4-AD to create the saxairtab yeast 2-hybrid library. To begin the procedure for creating yeast populations with bait and prey libraries, inoculate three milliliter cultures of each of the MATa yeast transformants carrying the various trip one containing bait plasmids and CSM minus trip medium.Include two separate cultures, containing the vector plasmid alone, to serve as procedural controls. Incubate the cultures at 30 degrees Celsius and 200 rpm for six hours. Then dilute each culture into a 25 milliliter culture in a sterile Erlenmeyer flask and incubate overnight.Thaw a frozen vile of MAT alpha cells containing the lu2-carrying prey library at room temperature. Use the entire thawed vile of cells to inoculate 125 milliliters of CSM minus lu minus met medium in a sterile Erlenmeyer flask. Grow the culture at 30 degrees Celsius and 200 rpm overnight.On the following day, after confirming that the OD600 of the overnight mat a transformant cultures range between 1.0 to 1.5, transfer the cultures to 50 milliliter conical tubes and centrifuge 21 OD 600 equivalents of each culture at 4, 696 times g at room temperature for five minutes. Resuspend each pellet of mat a cells in 10 milliliters of sterile water, transfer the suspension to a new 50 milliliter conical tube, and centrifuge at 4, 696 times g at room temperature for five minutes. Using a pipette, gently remove the supernatant, without disrupting the pelletted cells, and resuspend the pellet in four milliliters of buffered YPDA medium at pH 3.7.For every 10 mating reactions desired, pellet 39 OD 600 equivalents of the mat alpha strain carrying the library plasmids in a 50 milliliter conical tube. Resuspend the mat alpha cells in 10 milliliters of sterile water and repellet in a new 50 milliliter conical tube at 4, 696 times g at room temperature for five minutes. Using a pipette, gently remove the supernatant, without disrupting the pelletted cells, and resuspend the pellet in 10 milliliters of buffered YPDA medium at pH 3.7.To set up each mating reaction, add the following to a new 50 milliliter conical tube:One milliliter of MAT-A transformed cells, one milliliter of MAT-alpha library containing cells, and one milliliter of buffered YPDA at pH 3.7. Incubate at 30 degrees Celsius with gentle, orbital agitation for 90 minutes. After 90 minutes, centrifuge the cells at 4, 696 times g at room temperature for five minutes.Remove the supernatant and resuspend the pellet in two milliliters of one to one buffered YPDA-YPD. Plate all two milliliters of cells onto a 100 milliliter YPD plate and incubate at 30 degrees Celsius for approximately 20 hours. To harvest the cells, pipette two to three milliliters of CSM minus lu minus trip medium onto each YPD plate and use a cell scraper to dislodge the cells.Pipette the dislodged cells into a 50 milliliter conical tube. Rinse the plate four to five times with two to three milliliters of CSM minus lu minus trip medium by using a 1, 000 microliter pipette to pipette up the medium and gently ejecting it across the YPD plate surface. Centrifuge the cells at 4, 696 times g at room temperature for five minutes.Discard the supernatant from each conical tube and resuspend the cells in 40 milliliters of CSM minus lu minus trip medium by pipetting up and down. To estimate the number of diploid cells formed, dilute four microliters of the diploid mixture into 200 microliters and 2, 000 microliters of CSM minus lu minus trip medium, respectively. Plate 200 microliters of each dilution onto a CSM minus lu minus trip plate to represent a one to 10, 000 and one to 100, 000 full dilution of the stock of diploids harvested.Incubate the plates at 30 degrees Celsius for 36 to 40 hours. Immediately use the remainder of each 40 milliliters of cell resuspension to inoculate 500 milliliters of CSM minus lu minus trip medium in a one liter Erlenmeyer flask. Measure the OD 600 of each culture.Incubate these flasks at 30 degrees Celsius, with shaking at 180 rpm, until they reach saturation, which typically takes 36 to 40 hours. Monitor cell growth at 24 hours and 36 hours by measuring the OD 600. Before continuing with the procedure, confirm the mating efficiency of the two yeast strains.A minimum number of 200 colonies on the one to 10, 000 dilution plate is required. Once the cultures have reached saturation, use a pipette to remove a 20 milliliter alloquade from each culture and inoculate two liter Erlenmeyer flasks. One flask containing 750 milliliters of CSM minus lu minus trip medium and a second flask containing 750 milliliters of CSM minus lu minus trip minus his medium.With the lowest level of three amino 124 triasol that eliminates background, determined previously as described in the text protocol. Mix the new cultures well by swirling. After measuring the initial OD 600, incubate the cultures at 30 degrees Celsius, while shaking at 180 rpm, until saturation, which typically occurs within 24 hours for the unselected CSM minus lu minus trip culture, but can take over 70 hours for cultures under selection for yeast 2-hybrid interactions.Once cultures have reached saturation, remove 11 milliliters of each culture by pipette and sediment the cells with a five minute spin at 4, 696 times g at room temperature. Discard the supernatant and proceed with DNA extraction as demonstrated in the next section. To begin the DNA extraction procedure, use a pipette to resuspend each cell pellet prepared earlier in 500 microliters of strong TE buffer.Transfer to a 1.5 milliliter micro-centrifuge tube and add 10 microliters of zimolay stock to each tube. In a fume hood, add three microliters of beta marketol ethanol to each tube and mix well. Incubate in the 37 degree Celsius incubator for 24 to 36 hours.Next, extract the samples two times with 500 microliters of fenol chloroform isolaymal alcohol as described in the text protocol. Add seven microliters of four molar sodium chloride and 900 microliters of ice cold, 100%ethanol and mix by inversion. Sediment the DNA by spinning in a micro-centrifuge at 21, 130 times g for 10 minutes at room temperature.Discard the supernatant by pipetting and wash each pellet three times with 900 microliters of 70%ethanol. Sediment the DNA by centrifuging at 21, 130 times g for two minutes. After removing the residual ethanol, dry the pellets at 42 degrees Celsius for seven minutes.Resuspend each pellet in 120 microliters of 0.1x strong TE in a 37 degree Celsius water bath for 90 minutes. Pipette 60 microliters of the extracted DNA into a sterile 1.5 milliliter micro-centrifuge tube. Add 120 microliters of strong TE and 3.5 microliters of RN ace A stock, flick to mix, and incubate at 37 degrees Celsius for one hour.Add seven microliters of five molar ammonium acitate and 900 microliters of ice cold, 100%ethanol to each tube. Mix by inversion and sediment the DNA, as demonstrated previously. Resuspend the RNace-A treated DNA in 55 microliters of 0.1x strong TE in a 37 degree Celsius water bath for 90 minutes.Quantify the DNA by absorbance at 260 nanometers. The next step is to perform PCR on the CDNA inserts. Perform two 50 microliter PCR reactions per DNA sample.To each reaction add water, five micrograms of DNA sample, 25 picamoles of each forward and reverse primer matching the prey library plasmid, and 25 microliters of high fidelity 2x PCR master mix. Amplify the reactions with this program. A 30 second de-naturation at 98 degrees Celsius, 25 cycles of D-natrine at 98 degrees Celsius for 10 seconds, anealing at 55 degrees Celsius for 30 seconds, and extension times of three minutes at 72 degrees Celsius, followed by a five minute incubation at 72 degrees Celsius.Analyze each PCR reaction by 1%DNA agrose chill electrophoresis with a gel containing athidiumbromine and visualize the gel by UV transillumination. Samples without selection show a generalized smear of DNA, but upon selection individual bands can be discerned. Combine duplicate PCR samples and purify using a kit following the manufacturers instructions.Quantify the DNA by absorbance at 260 nanometers on a spectrophotometer. Subsequently, perform deep sequencing, followed by bioinformatics processing and verification as described in the text protocol. The new random fragmentised yeast 2-hybrid library was used in the demonstrated procedure and the plasmid inserts amplified by PCR were analyzed by DEEP sequencing and bioinformatics processing using the DEEPN software and compared with a previously published library.This plot shows the rank order of reeds per each gene in the libraries divided by reeds per gene found by sequencing the yeast genome. This histogram shows the number of unique plasmids per gene that encodes a fragment that is both in the protein coding region and in the proper translational reading frame. A comparison of the numbers of different plasmids in each library that encode yeast genes and the proportion that are and are not in the correct translational reading frame or are inserted backwards is shown.Overall, the new library has many more different plasmids. These methods ensure that the library can be reproducibly transferred to cells containing different bait proteins and that the selection procedure can yield reproducible sets of genes that are enriched, distinguishing them as specific candidate-interacting proteins. Once mastered, this technique can be done in about a week if performed properly.Most of this work is incubating cultures of cells and the actually hands-on time is just a few hours. While attempting this procedure, it's important to remember to create populations of a large number of individual diploid cells so that the library can be reproducibly transferred to cells containing the different bait proteins. It is also important to pay close attention to the dilution conditions and growth rate of cells for those populations undergoing selection for positive yeast 2-hybrid conditions.Following this procedure, computational methods that are explained in the accompanied work will identify the candidate interacting proteins and provide a path for validating these interactions using a traditional yeast 2-hybrid assay or other biochemical methods. After watching this video, you should have a very good idea about how to reproducibly create diploid yeast populations that contain the prey plasmid library and grow them in batch to select for positive yeast 2-hybrid interactions. Don't forget that working with organic solvents, during the DNA purification protocol, can be dangerous and should be done in the fume hood wearing gloves and a lab coat.

a-yeast-2-hybrid-screen-in-batch-to-compare-protein-interactions

Research

JoVE Journal

Biochemistry

13.0K vistas.  University of Iowa. This method uses the principle of the yeast 2-hybrid system in both populations to identify protein-protein interactions that answer key questions about a variety of molecular and cellular processes. This technique leverages deep sequencing to see the interactions of one protein versus another. Since this approach is geared to make parallel comparisons, it can identify sets of proteins that interact with one protein versus another or different confirmations of the same protein or while typing...