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 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.
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., 'prey' fusion protein) and a given 'bait' 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 'prey' 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 'baits' 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 'bait' fusion proteins comprised of the protein of interest and a DNA-binding domain are 2µ-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.
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 '+40Ade' specify a total of 40 mg/L adenine.
2. Cloning and Verification of Bait Plasmids
NOTE: Construction of Gal4-DNA-binding domain Plasmids. Currently, there are a variety of commercially available and academically available Y2H systems. DEEPN can accommodate many of these provided that the bait plasmid expressing the protein of interest fused to a DNA-binding domain is in a TRP1-containing plasmid. Other downstream requirements are that the sequence immediately upstream of the prey library insert is known and that a positive Y2H interaction can be scored by the production of His3 allowing for selection in media lacking histidine. Here we will describe use of a new Y2H bait plasmid (pTEF-GBD, Figure 2), however, other Y2H bait plasmids including pGBKT7 can be used as well. For construction and evaluation of bait plasmids, we will describe use of pTEF-GBD. As a general note, we recommend gene synthesis to produce an open-reading frame that adheres to the yeast codon bias to help ensure good expression and ease with cloning. Ensure that the cloning scheme allows for the bait to be in-frame with the Gal4 DNA-binding domain and that when cloning into the 3' site, a stop codon follows the bait-coding region.
3. Expression of Gal4-DNA-binding Domain Fusion Proteins
4. Self-activation Test
5. Create Yeast Populations with Bait and Prey Library
NOTE: The Y187 strain that houses commercial prey library plasmids does not mate well. Thus, the following optimized conditions are required to maintain complexity of the library. The PLY5725 strain containing Y2H prey libraries mates better and the same mating procedure can be used with this strain (Figure 5).
6. Sample preparation for DEEPN Deep Sequencing
7. Deep sequencing
NOTE: Sample preparation and sequencing on a deep sequencing platform is typically available in commercial and academic DNA sequencing core facilities.
8. Bioinformatic Processing and Verification
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 mating efficiency of the bait containing MATA yeast with the library-containing MATalpha yeast, and finally, low-stringency conditions that allow the population to grow under conditions that select for many positive Y2H reactions, even ones that produce low levels of His3 activity and a weak Y2H transcriptional response.
One of the critical aspects of DEEPN is to reproducibly introduce the Y2H library into strains that contain different bait plasmids and to reliably select those populations for those library plasmids that create positive Y2H interactions. This becomes more difficult when the complexity of the Y2H library increases since it is harder to ensure adequate transfer of the entire library across different initial populations. In addition, the size of populations chosen to grow under selection conditions and depth of sequencing have to be large enough to observe reproducible changes in the Y2H plasmid composition to reliably identify true positive Y2H interactions. In previous studies, we used commercially available Y2H cDNA libraries. We found the variability in the Y2H library distribution between separate populations carrying the same bait plasmids is low, with an overdispersion between the two initial populations before selection of <0.01 typically, and 0.35 – 0.55 for separate populations after selection4. However, the complexity of some of these commercially available Y2H libraries is fairly low (Figure 7). In addition, many of the clones within it (~60%) are made entirely of cDNA fragments that are 3' of the coding region, which further limits their utility. To demonstrate that the methods above were capable of accommodating more complex Y2H libraries, we created a new Y2H library in the streamlined 'prey' plasmid vector (pGal4AD) containing fragments of genomic DNA from Saccaromyces cerevisiae (strain PLY5725). Genomic DNA was fragmented by shearing, size selected for ranges of 600 – 1500 bp, modified with adaptors, and inserted into pGal4AD to create the SacCer_TAB Y2H library (Figure 8). The library was transformed into PLY5725, which produced a yeast population that was then mated to separate samples of PJ69-4A MATA carrying the pTEF-GBD plasmid alone. Duplicate diploid populations were grown under non-selective and selective conditions. The Y2H plasmid library inserts were analyzed by deep sequencing. When mapped to genomic DNA encompassing 100 bp upstream and downstream of each protein coding region (84% of the whole genome), we found >1.1 million different plasmids in the library for an estimated total size of the library in yeast of ~1.35 million different plasmids. As a comparison, we also transformed a previously described yeast genomic Y2H library11 (PJ_C1,C2,C3 Y2H library) in MATalpha yeast, and subjected it to the same analysis as above. We found that the complexity of our random fragment yeast Y2H library was far higher and had far more plasmids encoding in-frame fragments of each gene than the previously published library (Figure 9). Importantly, the generation of initial library containing diploid populations was very reproducible with an overdispersion of <0.01 (Figure 10). Moreover, the reproducibility of having the two separate yeast populations produce similar redistributions of plasmids after selecting for positive Y2H interactions was very good, yielding an overdispersion of 0.3. Thus, the methods here accommodate larger Y2H libraries of higher complexity than those previously used.
In terms of increasing the ease of DEEPN, we prefer using gene synthesis to make inserts coding for the bait protein of interest. This allows coding regions for protein domains, chimeras, and mutants to be easily incorporated into Y2H bait plasmids, but also allows their codons to be optimized for expression in S. cerevisiae. Expression of two different Gal4-DNA-binding domain fusion proteins is demonstrated in Figure 3. Two different yeast transformants expressing either of two bait fusion proteins were prepared and subjected to SDS-PAGE and immunoblotting with anti-myc antibodies. Note expression levels of bait proteins relative to the Gal4 DNA-binding domain alone from the empty vector. We found it is important not only to verify the expression of the bait fusion protein, but also to verify expression in the exact MATA yeast transformant colony that will be used to expand and mate to the prey library-containing yeast. Once a transformant has been identified, it is possible to freeze it down and store for later use.
Figure 4 shows a test for self-activation where a serial dilution of diploid cells are plated onto CSM-Leu-Trp (+His), CSM-Trp-Leu-His (-His), and CSM-Trp-Leu-His+3AT (-His+3AT) plates and allowed to grow at 30 °C for 3 days. The desired result is to observe growth in the presence but not absence of histidine regardless of whether there is 3AT. This will allow the use of CSM-Trp-Leu-His to select for yeast with a positive yeast 2-hybrid interaction. If there is growth on CSM-Leu-Trp-His plates, then a Y2H selection can still be obtained using the lowest concentration of 3AT that prevents growth. We find that if growth can be blocked in CSM-Trp-Leu-His + 0.1 mM 3AT, then the DEEPN assay can proceed using this condition to select for Y2H interactions. If, however, inhibition of the growth of diploids containing pGal4AD or other empty prey plasmid and the pTEF-GBD-bait fusion plasmid requires higher concentrations of 3AT, it will compromise the performance of the DEEPN procedure and a different bait plasmid should be sought. Note that His+ growth is the only selection for a positive Y2H interaction. We have found this is sufficient to enrich for Y2H interactions in batch.
One of the most critical procedures in the DEEPN workflow is to achieve high efficiency mating of the MATA yeast carrying the bait plasmid with the MATalpha yeast carrying the Y2H prey library. We found that some strains (e.g., Y187)18, housing commercially available cDNA libraries, have relatively poor mating efficiency. For that reason, we engineered a strain to carry Y2H libraries. This strain is based on BY4742, a derivative of S288c. This strain lacks GAL4, GAL80, TRP1, LEU2, and HIS3. It contains no reporters that are responsive to a hybrid Gal4 protein nor a different hybrid (e.g., LexA-VP16). Instead, the source of Y2H-induced His3 production is housed within the MATA strain that would carry the particular bait plasmid. This simplifies the system and allows greater flexibility in that one can use the same library-containing MATalpha cells to mate with a strain expressing a LexA-bait fusion protein and a LexA(UAS)-HIS3 reporter or a Gal4-DBD-bait fusion protein and a Gal4(UAS)-HIS3 reporter.
Once the diploid populations carrying both a bait plasmid and the library are generated, they are diluted and allowed to grow under conditions that just select for both plasmids (e.g., CSM-Trp-Leu) or for both plasmids and a positive Y2H interaction that drives production of His3 (e.g., CSM-Leu-Trp-His). It is important to start with a large amount of the starting population to avoid an evolutionary 'bottleneck' that can skew the population that results after selection for a positive Y2H interaction. Thus, our procedure specifies using 20 mL of out of the 500 mL of diploid population culture to be grown in 750 mL of fresh CSM-Leu-Trp-His media to avoid this problem. With the pTEF-GBD as the bait plasmid, we found that a single round of dilution and growth was sufficient to evolve an informative population. Using different bait plasmids previously, we used two rounds of dilution and growth, an initial 20 mL of into 750 mL, and then 2 mL of from that saturated culture diluted in 75 mL. Figure 6 shows the results from PCR amplification across the library inserts for the diploid population grown under non-selective conditions, as well as a first and second successive round of dilution and growth in selective condition for two different bait plasmids. Note that with no selection, there is a generalized smear of PCR products indicative of a complex and relatively well-normalized mixture of fragments. Upon selection, that pattern changes, with some species greatly enriching so that individual bands can be discerned. Some degree of banding is typical of the successful experiments conducted so far, yet a smear pattern in addition to, or instead of a banding pattern, is indicative of a complex mixture of prey inserts and is desirable if one wants to maximize the number of candidates that DEEPN detects. Very strong banding pattern, where most of the PCR product is found in 1 – 3 bands, indicates that most of sequencing data will be dominated by only 1 – 3 prey inserts. One can compensate for this partly by dedicating more reads from this sample. One can see that if the population is diluted and grown further (see 'selected d2' in Figure 5), the banding pattern is more prominent, indicating that prey plasmids conferring weak but authentic Y2H interactions are being diminished while a select few prey plasmids are increasing their abundance. Successive dilution and growth to this excessive level is deemed counterproductive to the goal of catching the largest number of potential candidates and thus with the protocol here, we recommend one round of dilution and growth be used.
Figure 1: Schematic of DEEPN workflow. The general outline of the laboratory procedures is shown on the left along with the approximate time needed to complete the corresponding task. On the right is the bioinformatics workflow using the DEEPN and Stat_Maker software packages. Please click here to view a larger version of this figure.
Figure 2: Schematic of pTEF-GBD. The TRP1-containing low copy Gal4 DNA-binding domain fusion protein expression plasmid is shown. It features a constitutive TEF1 promoter, myc epitope tag, the Gal4 DNA binding domain followed a T7 RNA polymerase binding site, polylinker, and PRM9 terminator within a centromere (CEN)-based plasmid. This plasmid also carries Kanamycin resistance and the ColE1 bacterial origin of replication. Please click here to view a larger version of this figure.
Figure 3: Expression of Gal4-bait fusion proteins. Lysates from yeast expressing different bait proteins fused to the Gal4 DNA-binding domain expressed in pTEF-GBD were subjected to SDS-PAGE and immunoblotting with anti-myc antibodies. pTEF-GBD expresses just the Gal4-DNA-bindng domain alone; pTEF-GBD-bait1 expresses the Gal4-DNA-bindng domain fused to a RhoA lacking its C-terminal prenylation site and housing a mutation locking it into a GTP-bound conformation; pTEF-GBD-bait2 expresses the Gal4-DNA-bindng domain fused to a RhoA lacking its C-terminal prenylation site and housing a mutation locking it into a GDP-bound conformation. Please click here to view a larger version of this figure.
Figure 4: Self-Activation Test. Diploids made from PJ69-4A cells carrying the indicated bait plasmids and PLY5725 cells carrying the indicated prey plasmid were serially diluted and spotted onto CSM-Leu-Trp plates, CSM-Leu-Trp-His plates, and CSM-Leu-Trp-His+3AT plates and grown for 3 days at 30 °C. The PJ69-4A transformants used for mating were the first of each pair shown in Figure 3. Please click here to view a larger version of this figure.
Figure 5: Mating efficiency of Y187 vs. PLY5725. The mating reaction between PJ69-4A containing a TRP1-containing bait vector and Y187 or PLY5725 carrying prey plasmid was performed. 1:10,000 dilutions were plated onto CSM-Trp-Leu to select for diploids. The PLY5725 strain shows a higher mating efficiency than the Y187 strain with ~10 fold more colonies produced. Please click here to view a larger version of this figure.
Figure 6: PCR of prey library inserts. DNA was isolated from diploid yeast containing a prey library that was grown under non-selective conditions (CSM-Leu-Trp media) or conditions selecting for a positive Y2H interaction (CSM-Leu-Trp-His media). PCR across the library inserts reveals differences in the repertoire of inserts selected. Two rounds of selective growth were used. An initial round of growth made by diluting 20 mL of the 500 mL of diploid culture into 750 mL of non-selective CSM-Leu-Trp media, and another 20 mL of into 750 mL of CSM-Leu-Trp-His media for an initial round of selective growth (d1). This was followed by an additional round of growth (d2) made by taking 2 mL of the d1 culture and diluting into 75 mL of selective media CSM-Leu-Trp-His. Please click here to view a larger version of this figure.
Figure 7: Complexity of Y2H cDNA library. Analysis of the content of a commercially available mouse cDNA Y2H prey libraries: a library from cDNA of mouse brain and one from multiple mouse tissues. Please click here to view a larger version of this figure.
Figure 8: Generation of Y2H yeast genomic fragment library. A. Schematic describing assembly of the yeast genomic fragment library into pGal4AD. Genomic DNA from strain PLY5725 was randomly sheared, ligated with the indicated Y-adaptors, and ligated into SfiI-cut pGal4AD. Ligations were transformed into bacteria to yield 2.2 x 106 independent colonies that were combined and grown prior to isolation of their plasmid DNA to comprise the SacCer_TAB Y2H library. B. The LEU2-containing plasmid (pGal4AD) housing the Gal4 transcriptional activation domain is shown. Please click here to view a larger version of this figure.
Figure 9: Complexity of Y2H yeast genomic library. The SacCer_TAB genomic library was transformed into the PLY5725 MATalpha strain and the PJ_C1,C2,C3 yeast genomic library was transformed into the Υ187 ΜΑΤalpha strain. Diploids of these populations were made by mating to the PJY69-4A strain. Populations were grown for 10 generations and prey fragments PCR amplified were subjected to high-throughput sequencing and analysis. A. Shows the rank order of reads per each gene in the Y2H libraries divided by the reads per gene found by sequencing the yeast genome. Given equivalent abundance of each gene, each gene would have a value of 1. B. Histogram showing the number of unique plasmids per gene that encode a fragment that is both in the protein coding region (ORF) and in the proper translational reading frame. C. Comparison of the number of different plasmids in each library that encode yeast genes and the proportion of these that are and are not in the correct translational reading frame or are inserted backwards. D. Plots showing positions and abundance of the junctions that map to example genes (VPS8 and VPS16) found for the plasmids in each library. Plasmids with gene fusions that are in the correct translational reading frame are designated blue. Please click here to view a larger version of this figure.
Figure 10: Reproducibility of DEEPN with complex Y2H yeast genomic library. A. Four different yeast diploid populations were created by mating with the SacCer_TAB Y2H library housed in PLY5725, grown under non-selective conditions, and sequenced. The reads per gene for each population individually is plotted as a function of the average rank order value across all populations. B. Shows the reads per gene between two samples after selection for positive Y2H interactions. Please click here to view a larger version of this figure.
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 accessible to most laboratories equipped for standard molecular biology. DEEPN allows selection from the same prey library population while using different bait plasmids. Hence, the set of interacting candidates that produce a Y2H interaction with one bait vs. another can be directly compared. Because deep sequencing is used, one can verify the starting library composition for each bait-specific yeast population and follow the enrichment of each candidate prey gene in the library independently. Batch processing allows querying the same library against different baits in a semi-quantitative manner that then allows one to calculate a statistical ranking4.
There are several steps in this protocol that are critical for success. One is that a large number of diploids must be obtained to ensure adequate representation of the Y2H prey library is mixed with each bait plasmid of interest. The mating procedure described here has been optimized by varying several parameters. We found that some strains that house commercial Y2H libraries mate poorly in general, however, the mating procedure described here can generate 2 x 106– 2 x 107 diploids when followed, allowing adequate and reproducible transfer of the Y2H library into the population. Another critical aspect of the procedure is to ensure that the bait plasmid of interest does not create a positive Y2H interaction on its own or with the empty Gal4 activation domain prey plasmid. The method for selecting a Y2H interaction is demanding growth in the absence of Histidine wherein a positive Y2H interaction induces transcription of HIS3 to allow cells to be His+. While routinely the traditional Y2H assays can add the competitive inhibitor 3AT to diminish background growth or use other reporters3 , this procedure works best when cells with weak Y2H interactions can grow and thus increasing the stringency for growth and selecting only for cells with strong Y2H interactions limits the repertoire of prey plasmids that can be identified. Another critical aspect is to make sure that a large amount of the starting diploid population used to grow under selective and non-selective conditions to avoid evolutionary bottlenecks from sampling error4 . Following the culture volumes and cell numbers specified here will help ensure reproducibility and diminish noise.
One of the reasons the DEEPN approach is powerful is that it can comprehensively follow all the plasmids in a given prey library for their ability to interact with the bait of interest. Thus, one of the limitations of DEEPN is the complexity of the library used. For some of the commercial libraries like the ones we used here, we found that the number of plasmids that encode bona fide fragments of cDNA ORF that are in the same reading frame of Gal4 activation domain is between 3 – 6 x 104 with representation of ~6,000 – 8,000 different genes. We found that nearly 75% of these libraries contained fragments corresponding solely to cDNA regions that were 3' of the coding DNA sequence (CDS/ORF). Moreover, almost a third of the genes which had fragments in the library had none that corresponded to regions in the ORF or upstream of the ORF.
We made three other changes to the DEEPN method. One is a new MATalpha strain that can carry 'prey' libraries housed within a TRP1 plasmid and that mates far better than some commercially available library-containing strains such as Y187. This helps ensure complete transfer of the library population for each bait of interest. We also made a new 'bait' expression plasmid, which differs from previously described plasmids that use a variable copy 2µ-based backbone10. Here we describe a low-copy CEN plasmid (pTEF-GBD) and find that a single round of selective growth is sufficient to enrich for interacting prey plasmids. Lastly, we build a streamlined 'prey' library vector and used to produce a high-density random-fragment genomic Y2H library for Saccharomces cerevisiae, which should be helpful in the future for discovering and characterizing interactions amonst yeast proteins. Overall, the improvements in materials and bioinformatics tools (described in the accompanying work) make the DEEPN approach an accessible, feasible, and efficient way of performing comprehensive and comparative Y2H screens.
The authors have nothing to disclose.
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.
Illumina HiSeq 4000 | Illumina | deep sequencing platform | |
Monoclonal anti-HA antibodies | Biolegend | 901514 | Primary Antibody to detect expression of HA in pGal4AD constructs |
Polyclonal anti-myc antibodies | QED Biosciences Inc | 18826 | Primary Antibody to detect expression of MYC in pTEF-GBD constructs |
NarI | New England BioLabs | R0191S | |
EcoRI-HF | New England BioLabs | R3101S | |
BamHI-HF | New England BioLabs | R3236S | |
XhoI | New England BioLabs | R0146S | |
Polyethylene Glycol 3350, powder | J.T. Baker | U2211-08 | |
Salmon Sperm DNA | Trevigen, Inc sold by Fisher Scientific | 50-948-286 | carrier DNA for yeast transformation section 3.2.1. |
Kanamycin Monosulfate | Research Products International | K22000 | |
LE Agarose | GeneMate | E-3120-500 | used for making DNA agarose gels |
Sodium Chloride | Research Products International | S23025 | |
Tryptone | Research Products International | T60060 | |
D-Sorbitol | Research Products International | S23080 | |
Lithium Acetate Dihydrate | MP Biomedicals | 155256 | |
Calcium Chloride | ThermoFisher | C79 | |
EDTA Sodium Salt | Research Products International | E57020 | |
Yeast Extract Powder | Research Products International | Y20020 | |
Yeast Nitrogen Base (ammonium sulfate) w/o amino acids | Research Products International | Y20040 | |
CSM-Trp-Leu+40ADE | Formedium | DCS0789 | |
CSM-Trp-Leu-His+40ADE | Formedium | DCS1169 | |
CSM-Leu-Met | Formedium | DCS0549 | |
CSM-Trp-Met | Bio 101, Inc | 4520-922 | |
L-Methionine | Formedium | DOC0168 | |
Adenine | Research Products International | A11500 | |
D-(+)-Glucose | Research Products International | G32045 | |
Bacto Agar | BD | 214010 | used for making media plates in section 1 |
Peptone | Research Products International | P20240 | |
3-amino-1,2,4 Triazole | Sigma | A8056 | |
2-Mercaptoehanol (BME) | Sigma-Aldrich | M6250 | |
Zymolyase 100T | USBiological | Z1004 | |
Potassium phosphate dibasic | Sigma | P8281 | |
Phenol:Chloroform:IAA | Ambion | AM9732 | |
Ammonium Acetate | Sigma-Aldrich | 238074 | |
Ethanol | Decon Laboratories, INC | 2716 | |
RNAse A | ThermoFisher | EN0531 | |
Urea | Research Products International | U20200 | |
SDS | Research Products International | L22010 | |
glycerol | Sigma Aldrich | G5516 | |
Tris-HCl | Gibco | 15506-017 | |
bromophenol blue | Amresco | 449 | |
Gibson Assembly Cloning Kit | New England Biolabs | E5510S | Rapid assembly method for cloning of plasmids in section 2 |
NEBNext High-Fidelity 2x PCR Master Mix | New England Biolabs | M0541S | 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 |
Ethidium Bromide | Amresco | 0492-5G | |
QIAquick PCR purification kit | Qiagen | 28104 | Used for purification of pcr products in section 6.2.3 |
Qiaquick DNA Gel Extraction Kit | Qiagen | 28704 | Used for purification of digested pTEF-GBD in section 2.1 |
KAPA Hyper Prep kit | KAPA Biosystems | KK8500 | preparation kit for deep sequencing |
Codon optimization | http://www.jcat.de | ||
Codon optimization | https://www.idtdna.com/CodonOpt | ||
gBlocks | Integrated DNA Technologies | DNA fragments used for cloning in Section 2.2 | |
Strings | Thermofisher | DNA fragments used for cloning in Section 2.2 | |
GenCatch Plasmid DNA mini-prep Kit | EPOCH Life Sciences | Used to prepare quantities of DNA in Section 2.3 | |
Covaris E220 | Covaris | high performance ultra-sonicator in section 7 | |
oligo nucelotide 5’- CGGTCTT CAATTTCTCAAGTTTCAG -3’ |
Integrated DNA Technologies or Thermofisher | used for pcr amplification and sequencing 5' insert pTEF-GBD during plasmid construction | |
oligo nucelotide 5’-GAGTAACG ACATTCCCAGTTGTTC-3’ |
Integrated DNA Technologies or Thermofisher | used for pcr amplification and sequencing 5' insert pTEF-GBD during plasmid construction | |
oligo nucelotide 5’-CACCGTAT TTCTGCCACCTCTTCC-3’ |
Integrated DNA Technologies or Thermofisher | used for pcr amplification and sequencing 3' insert pTEF-GBD during plasmid construction | |
oligo nucelotide 5’-GCAACCGC ACTATTTGGAGCGCTG-3’ |
Integrated DNA Technologies or Thermofisher | used for pcr amplification and sequencing 3' insert pTEF-GBD during plasmid construction | |
oligonucleotide 5’-GTTCCGATG CCTCTGCGAGTG-3’ |
Integrated DNA Technologies or Thermofisher | 5' Pimer used for insert amplification of pGAL4AD | |
oligonucelotide 5’-GCACATGCT AGCGTCAAATACC-3’ |
Integrated DNA Technologies or Thermofisher | 3' Pimer used for insert amplification of pGAL4AD | |
oligonucelotide 5’-ACCCAAGCA GTGGTATCAACG-3’ |
Integrated DNA Technologies or Thermofisher | 5' Pimer used for insert amplification of pGADT7 | |
oligonucelotide 5’- TATTTAGA AGTGTCAACAACGTA -3’ |
Integrated DNA Technologies or Thermofisher | 3' Pimer used for insert amplification of pGADT7 | |
PJ69-4A MatA yeast strain | 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 | MATA leu2-3,112 ura3-52 trp1-901 his3-200 gal4D, gal80D, GAL-ADE2 lys2::GAL1-HIS3 met2::GAL7 | |
pTEF-GBD | Dr. Robert Piper Lab | Gal4-DNA binding doimain expression plasmid | |
PLY5725 MATalpha yeast strain | Dr. Robert Piper Lab | MATalpha his3∆1 leu2∆0 lys2∆0 ura3∆0 gal4∆ trp1∆ Gal80∆ | |
pGal4AD (pPL6343) | Dr. Robert Piper Lab | Gal4-activation domain expression plasmid | |
100 mm petri dishes | Kord-Vallmark sold by VWR | 2900 | |
125 mL PYREX Erlenmeyer flask | Fisher Scientific | S63270 | |
250 mL PYREX Erlenmeyer flask | Fisher Scientific | S63271 | |
1,000 mL PYREX Erlenmeyer flask | Fisher Scientific | S63274 | |
2,000 mL PYREX Erlenmeyer flask | Fisher Scientific | S63275 | |
20 X 150 mm Disposable Culture Tube | Thermofisher | 14-961-33 | |
pipet-aid | Drummond | 4-000-100 | |
5 mL Serological Pipette | Denville | P7127 | |
10 mL Serological Pipette | Denville | P7128 | |
25 mL Serological Pipette | Denville | P7129 | |
1,000 mL PYREX Griffin Beaker | Fisher Scientific | 02-540P | |
1,000 mL PYREX Reuasable Media Storage Bottle | Fisher Scientific | 06-414-1D | |
1,000 mL graduated cylinder | Fisher Scientific | 08-572-6G | |
SpectraMax 190 | Molecular Devices | used to measure the Optical Density of cells | |
NanoDrop 2000 | Thermo Scientific | ND-2000 | Spectrophotometer used to quantify amount of DNA |
Electronic UV transilluminator | Ultra Lum | MEB 20 | used to visualize DNA in an Ethidium Bromide agarose gel |
P1000 Gilson PIPETMAN | Fisher Scientific | F123602G | |
P200 Gilson PIPETMAN | Fisher Scientific | F123601G | |
P20 Gilson PIPETMAN | Fisher Scientific | F123600G | |
P10 Gilson PIPETMAN | Fisher Scientific | F144802G | |
1250 µL Low Retention Pipette Tips | GeneMate | P-1236-1250 | |
200 µLLow Retention Pipette Tips | VWR | 10017-044 | |
10 µL XL Low Retention Pipette Tips | VWR | 10017-042 | |
50 mL conical tube | VWR | 490001-627 | |
15 mL conical tube | VWR | 490001-621 | |
cell scraper | Denville Scientific | TC9310 | |
1.5 mL Microcentrifuge tubes | USA Scientific | 1615-5500 | |
HCl | Fluka Analytical | 318949-1L | |
NaOH | J.T. Baker | 5674-02 | |
Wooden applicators | Solon Care | 55900 | |
Eppendorf microcentrifuge 5424 | Fisher Scientific | 05-400-005 | microcentrifuge |
Sorvall ST16R | Thermo Fisher Scientific | 75004381 | benchtop centrifuge |
Amersham ECL Rabbit IgG, HRP-linked whole Ab (from donkey) | GE Healthcare | NA934-1ML | Secondary Antibody |
Amersham ECL Mouse IgG, HRP-linked whole Ab (from sheep) | GE Healthcare | NA931-1ML | Secondary Antibody |
SuperSignal West Pico Chemiluminescent Substrate | Thermo Fisher Scientific | 34080 | ECL detection solution |
Isotemp Incubator | Thermo Fisher Scientific | Incubator | |
Mutitron 2 | INFORS HT | Shaking incubator | |
Isotemp Digital-Control Water Bath Model 205 | Fisher Scientific | water bath | |
Y2H mouse cDNA library in Y187 (pan tissue) | Clontech | 630482 | commercially available cDNA Library |
Y2H mouse cDNA library in Y187 (mouse brain) | Clontech | 630488 | commercially available cDNA Library |
pGADT7 AD Vector | Clontech | 630442 | commercially available AD Vector housing many cDNA libraries |
pGBKT7 DNA-BD Vector | Clontech | 630443 | commercially available DNA-BD Vector |
Biolase DNA Polymerase | Bioline | BIO-21042 | DNA polymerase used for section 2.4 |
GeneMate GCL-60 Thermal Cycler | BioExpress | P-6050-60 | pcr machine |
TempAssure 0.5 mL PCR tubes | USA Scientific | 1405-8100 |