We are thankful for the thoughtful discussions with members of the Ju laboratory and Zhong laboratory, and the financial support from the Wright State University.

NOTE: The protocol described here is designed for screening library plasmids contained in ten 96-well plates but can be scaled up or down accordingly. The protocol needs to be repeated to complete the whole library screening. Usually, screening against 10 plates of library genes each time can be comfortably handled by 1 person.
1. Preparation for 96-well Yeast Transformation
NOTE: This step is done as previously described7,10.
<ol>
	<li>Aliquot 5 &#181;L (about 50&#8211;100 ng) of plasmid DNA from an arrayed plasmid library into each well of a round-bottom 96-well plate. 
	NOTE: An example of such a library would be a yeast gene overexpression library10.</li>
	<li>Inoculate 150 mL of yeast peptone dextrose (YPD) media in a 500 mL flask with a colony (2&#8211;3 mm diameter) of the haploid yeast strain W303&#945;. Incubate them overnight at 30 &#176;C with shaking (200 rpm).</li>
	<li>The following morning, measure the OD600 of the overnight culture and dilute the yeast culture to OD600 = 0.1 in (up to) 2 L of YPD. Incubate it at 30 &#176;C with shaking (200 rpm) for ~5 h until the culture reaches OD600 = 0.4&#8211;0.6.</li>
</ol>
2. Yeast Transformation
<ol>
	<li>Harvest the yeast culture by filling 8 sterile 250 mL centrifuge bottles and centrifuging them at room temperature at 3,000 x g for 10 min. Pour off the supernatant without disrupting the pellet. 
	NOTE: Carry out all centrifugation steps at room temperature.</li>
	<li>Wash the yeast with sterile-distilled H2O. For this, add 100 mL of sterile H2O to each centrifuge bottle and vortex it to resuspend the cell pellet. Combine the washed cells into 2 bottles and centrifuge them at 3,000 x g for 5 min. Pour off the supernatant.</li>
	<li>Wash the cells in each bottle in 100 mL of 0.1M LiOAc/1XTE (100 mM LiOAc; 10 mM Tris, pH 8.0; 1 mM EDTA) and centrifuge them at 3,000 x g for 5 min. Pour off the supernatant.</li>
	<li>While centrifuging, boil 5 mL of salmon sperm DNA (10 mg/mL) at 100 &#176;C using a block heater for 3 min and then cool it on ice.</li>
	<li>Resuspend the cell pellet in 25 mL of 0.1M LiOAc/1XTE in each bottle, combine the resuspended cells and transfer them to a 150 mL flask. Add 5 mL of the pre-cooled salmon sperm DNA and incubate it at 30 &#176;C with shaking (225 rpm) for 30 min.</li>
	<li>Pour the cell mixture into a sterile disposable reagent reservoir and, using a multichannel pipette, transfer 35 &#181;L of the cell mixture to each well of the round-bottom 96-well plate containing the library plasmid DNA. Vortex the 96-well plates using a plate vortexer for 1 min at 1,000 rpm. Incubate the plates for 30 min at 30 &#176;C without shaking. 
	NOTE: Do not stack the plates, so the heat can transfer more efficiently. We have found that vortexing the 96-well plates at 1,000 rpm does not cause liquid to spill out of the wells, but a safe vortex speed should be tested before performing the step.</li>
	<li>In a flask, prepare 200 mL of the transformation buffer containing a final concentration of 40% PEG3350, 10% DMSO, and 0.1M LiOAc. Prepare the transformation buffer immediately before use and mix it thoroughly by shaking.</li>
	<li>Remove 96-well plates from the 30 &#176;C incubator and mix them for 30 s at 1,000 rpm using the plate vortexer. Add 125 &#181;L of the transformation buffer to each well and then vortex the plates for 1 min at 1,000 rpm.</li>
	<li>Incubate the plates at 30 &#176;C for 30 min and then heat shock the yeast by placing the plates in a 42 &#176;C incubator for 15 min. 
	NOTE: Do not stack the plates.</li>
	<li>Centrifuge the plates for 5 min at 3,000 x g. Remove the transformation buffer from the wells by inverting the plates over a waste bin and forcefully dumping the buffer from the plates. Quickly blot the inverted plates on a clean paper towel to remove any liquid on the top of the plates.</li>
	<li>Rinse the cells by adding to the plates 200 &#181;L of minimal dropout medium corresponding to the selectable marker on the library plasmid. 
	NOTE: We used a synthetic Ura- medium.</li>
	<li>Centrifuge the plates for 5 min at 3,000 x g and remove the supernatant over a waste bin as in step 2.10.</li>
	<li>Add 160 &#181;L of minimal Ura-medium containing 2% glucose. Vortex the plates for 1 min at 1,000 rpm and incubate them at 30 &#176;C for 48 h without shaking. After 48 h, note that a pellet of transformed yeast is visible at the bottom of each well.</li>
	<li>Using a liquid dispenser, add 100 &#181;L of Ura-media containing glucose into each well of a new set of 96-well plates.</li>
	<li>Vortex the plates containing the transformed yeast (from step 2.13) at 1,000 rpm for 30 s. Using a sterile plastic 96-pin replicator, place the pins into the wells containing the transformed yeast and then inoculate them into the corresponding wells of the new plates filled with media. Incubate the new plates at 30 &#176;C for 24 h. 
	NOTE: If working with 10 plates, the medium can also be dispensed manually using multi-channel pipettes.</li>
	<li>After 24 h, a pellet of yeast should be visible in the bottom of each well containing successfully transformed yeast. To save these yeast strains as glycerol stocks, add 50 &#181;L of 50% glycerol to each well, vortex the plates for 30 s at 1,000 rpm, seal the plates with sealing tape and freeze them at -80 &#176;C. 
	NOTE: The above steps only need to be performed once. Future screenings of different yeast models against the same library disposable reagent reservoir can start from the glycerol stocks and proceed immediately from the steps below.</li>
</ol>
3. Mating Between Cells Containing Library Genes and Query Yeast
<ol>
	<li>To revive the library strains from the glycerol stock, take the 96-well plates out of the -80 &#176;C freezer, remove the sealing tape, and let the yeast thaw at room temperature for approximately 30 min.</li>
	<li>Once the yeast has thawed, use a sterile plastic 96-pin replicator to inoculate the glycerol stocks to 160 &#181;L of fresh Ura-media containing 2% glucose in 96-well plates and incubate them at 30 &#176;C for 24 h. Immediately after the library strains are used, seal the plates with sealing tape, and return them to the -80 &#176;C freezer.</li>
	<li>On the same day the library strains (W303&#945;) are thawed, inoculate the query yeast strain (here, FUS in W303a) to 50 mL of YPD and grow it overnight at 30 &#176;C with shaking at 250 rpm.</li>
	<li>The next morning, pour the query yeast strain to a sterile disposable reagent reservoir, and aliquot 160 &#181;L of the query strain to each well of a 96-well plate using a multichannel pipette.</li>
	<li>Using the liquid dispenser, dispense 160 &#181;L of YPD media into each well of ten 96-well plates. Use these plates later for mating the query yeast strain with the library yeast.</li>
	<li>Briefly vortex the 96-well plate containing the query strain and then use a sterile 96-pin replicator to transfer the query strain to the YPD plates.</li>
	<li>Briefly vortex the library strain plates and, for each library strain plate, use a new sterile 96-pin replicator to transfer the library strains to the YPD plates that have been inoculated with the query strain. 
	NOTE: Cells in glycerol lose their viability after multiple thaw-freeze cycles. Returning the library strains to the long-term storage (-80 &#176;C freezer) as soon as possible will keep the library in good quality, and ready for future use. Properly maintained, the glycerol stock can be frozen and thawed at least 10 times. We also strongly recommend keeping a working copy and backup copies of the glycerol stock. When the working stock does not work, immediately make a new working copy from the backup copy.</li>
	<li>Incubate the YPD plates at 30 &#176;C for 24 h. Note that a pellet of yeast will be visible in the bottom of each well after 24 h.</li>
	<li>Fill 96-well plates with a minimal dropout medium containing 2% raffinose, corresponding to the selectable markers on the plasmid in the query strain as well as on the library plasmid (e.g., here Ura-His-).</li>
	<li>Use a sterile 96-pin replicator to transfer yeast from the YPD mating cultures to the selective media. Incubate the 96-well plates at 30 &#176;C for 48 h; only yeast cells that have mated and formed diploid cells and, therefore, contain both the query plasmid and the library plasmid will be able to grow in this media. After 2 days, observe that a pellet is visible on the bottom of the wells.</li>
</ol>
4. Spotting Assay
<ol>
	<li>After 48 h of growth in the raffinose-containing selective media, spot the yeast on agar plates.
	<ol>
		<li>Prepare 2 sets of Ura-His- dropout plates containing 2% agar using clear polystyrene plates, one containing 2% galactose and the other containing 2% glucose.</li>
		<li>Vortex the 96-well plates for 1 min at 1,000 rpm, then spot the yeast to the Ura-His- dropout plates containing 2% galactose and 2% agar (FUS and library genes are induced) and to the Ura-His- dropout plates containing 2% glucose and 2% agar (FUS and library genes repressed) using a robotic spotting machine, by which the culture in each well is spotted onto the agar plates in quadruplicate (i.e., the culture in 1 well is spotted to 4 spots on the agar plate).</li>
		<li>After spotting, let the agar plates dry, and then place them upside down in a 30 &#176;C incubator. Photograph the agar plates every 24 h to record a faster/more yeast growth in which the toxicity to the query strain is rescued or to record a slower/less growth in which the toxicity to the query strain is exacerbated. Incubate the plates for 4 days. 
		NOTE: The culture in each well can be spotted on agar plates 1-to-1, as shown in a previous transformation-based method10. However, the 1-to-4 spotting here (which can be conveniently set up using the robotic spotting machine) significantly increases the robustness of the assay by reducing the number of false positives. Positive hits are only considered when all 4 colonies from the same well show a similar phenotype.</li>
	</ol>
	</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+genes+that+enhance+the+toxicity+of+a+mutant+huntingtin+fragment+or+alpha-synuclein.">Yeast genes that enhance the toxicity of a mutant huntingtin fragment or alpha-synuclein.</a> Science. 302 (5651), 1769-1772 (2003).</li><li>Outeiro, T. F., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+cells+provide+insight+into+alpha-synuclein+biology+and+pathobiology.">Yeast cells provide insight into alpha-synuclein biology and pathobiology.</a> Science. 302 (5651), 1772-1775 (2003).</li><li>Cooper, A. 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=Alpha-synuclein+blocks+ER-Golgi+traffic+and+Rab1+rescues+neuron+loss+in+Parkinson's+models.">Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models.</a> Science. 313 (5785), 324-328 (2006).</li><li>Treusch, S., 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=Functional+links+between+Abeta+toxicity,+endocytic+trafficking,+and+Alzheimer's+disease+risk+factors+in+yeast.">Functional links between Abeta toxicity, endocytic trafficking, and Alzheimer's disease risk factors in yeast.</a> Science. 334 (6060), 1241-1245 (2011).</li><li>Ju, S., 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+yeast+model+of+FUS/TLS-dependent+cytotoxicity.">A yeast model of FUS/TLS-dependent cytotoxicity.</a> PLoS Biology. 9 (4), 1001052 (2011).</li><li>Sun, Z., 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=Molecular+determinants+and+genetic+modifiers+of+aggregation+and+toxicity+for+the+ALS+disease+protein+FUS/TLS.">Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS/TLS.</a> PLoS Biology. 9 (4), 1000614 (2011).</li><li>Johnson, B. 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The authors have nothing to disclose.

Here, we describe a protocol to perform a plasmid overexpression screen in yeast using mating to introduce the plasmid library into the yeast model. Using this approach, multiple yeast models of neurodegenerative disease protein toxicity can be screened using the same collection of yeast transformed with a plasmid library. The laborious process of transformation only needs to be performed once, after which highly efficient yeast mating is used to introduce the plasmid library into the query strain. This protocol does rel...

The budding yeast Saccharomyces cerevisiae has been widely used in basic scientific research1 to understand cellular processes directly related to human diseases. Moreover, it has been used as a model organism for studying human disease-associated proteins, such as those linked to the most common neurodegenerative diseases, including Alzheimer&#39;s disease, Parkinson&#39;s disease, Huntington&#39;s disease, and Amyotrophic Lateral Sclerosis (ALS)2. An advantage of the yeast model is the ease with which a genome-wide screen can be performed to identify cellular pathways related to the toxicity of disease-related proteins, thus giving insight into the mechanism of their toxicity. One such screen is called an overexpression library screen, in&#160;which each of the 5,500 yeast genes in an arrayed library is transformed into a yeast model to identify which genes can modify toxicity when overexpressed. This screening method has been successfully applied in the yeast models of multiple neurodegenerative disease-associated proteins, including huntingtin for Huntington&#39;s disease3, &#945;-synuclein for Parkinson&#39;s disease4,5, A&#946; for Alzheimer&#39;s disease6, and FUS and TDP-43 for ALS7,8,9. While it is usually done in a high-throughput manner10, the most labor-intensive step of the screen is individually transforming 5,500 yeast genes from an arrayed library. This step must be performed each time the screening is repeated, and whenever a newly established yeast model needs to be studied. It is important to find a more efficient way to accomplish this task.
Yeast cells can stably exist in both haploid and diploid forms. There are two opposite mating types of haploid cells, mating type a and &#945;. Haploid cells of each mating type produce and secrete their own specific mating pheromone, to which only the opposite mating type cells respond. This allows mating between a and &#945; cells to produce stable diploid cells, a/&#945;. This process is spontaneous and highly efficient11. We can take advantage of this unique life cycle of S. cerevisiae to introduce the plasmid library. More specifically, each gene in the arrayed plasmid library will be transformed into haploid cells of one mating type, i.e., &#945; cell. These cells containing the library genes will then be stored in glycerol stock in an arrayed 96-well format. For each yeast model that needs to be screened, yeast cells containing the library genes can be thawed from the glycerol stock, and the screening can be done through&#160;mating with the yeast model of interest in the opposite mating type, i.e., mating type a. This idea of using mating to bring together two genes into yeast is not new. It has been successfully applied in the high-throughput yeast two-hybrid screening, in which a bait construct (i.e., Gal4-DNA-binding domain fusions) in one mating type is brought together through mating with a prey construct from an arrayed library12. However, this strategy has never been applied in&#160;overexpression library screenings, which have always used traditional transformation methods.
Our laboratory previously established a yeast model of the ALS-associated protein FUS7. Through overexpression library screening using the transformation method we discovered&#160;five yeast genes (ECM32, NAM8, SBP1, SKO1, and VHR1) that rescue toxicity of FUS when overexpressed. These findings were independently confirmed with a similar study by another group8. hUPF1, a human homolog of ECM32, was later shown to suppress toxicity in primary neuronal cells13&#160;and in an animal model of ALS14 as well. Using these five genes as proof of principle, we demonstrate that all five genes similarly rescue FUS toxicity when they are introduced into the FUS yeast model by mating. Since yeast cells containing the library genes can be stored permanently in glycerol stock and revived whenever needed, this mating-based method will remove the time-consuming step of transformation each time the library needs to be screened against. Since mating is highly efficient with no plasmid transformation involved, this strategy also significantly decreases the cost associated with purification and transformation of a large plasmid library. We will successfully apply this method to a library screening against yeast model of FUS.
The procedure for mating-based screening is briefly described in Figure 1. Initially, the arrayed plasmid library is transformed into a haploid yeast strain of mating type &#945; using a high-throughput yeast transformation protocol in which each well of a 96-well plate contains yeast transformed with a specific library plasmid. This collection of transformed yeast is saved as a glycerol stock that can be thawed and revived for use later on. The yeast model of interest, in&#160;this case FUS toxicity, must be generated in a haploid yeast strain with the opposite mating type (mating type a). In a high-throughput manner using sterile 96-pin replicators, the FUS strain and yeast strains containing the plasmid library are transferred to 96-well plates containing rich media and allowed to mate. Following&#160;mating, a small volume from each well of the mating culture is transferred to 96-well plates containing synthetic dropout media in which only diploid yeast containing both the FUS and library genes can grow. A robotic spotting machine is then used to transfer yeast culture from each well onto agar plates where the expression of FUS and the library genes is induced.&#160; Additionally, yeast culture is spotted&#160;to control agar plates where FUS and the library genes are not expressed. Following&#160;growth on&#160;agar plates, genes that rescue or exacerbate FUS toxicity will be identified.

<table border="0" cellpadding="0" cellspacing="0" style="width:555px;" width="554">
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			<td height="42" style="height:42px;width:183px;">salmon Sperm DNA (SS-DNA)</td>
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			<td height="84" style="height:84px;width:183px;">YPD broth</td>
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			<td style="width:99px;">Y20090</td>
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			<td height="21" style="height:21px;width:183px;">Granulated Agar</td>
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			<td style="width:99px;">G32040</td>
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			<td style="width:99px;">G33000</td>
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			<td height="84" style="height:84px;width:183px;">D-(+)-Raffinose Pentahydrate</td>
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			<td style="width:99px;">R20500</td>
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			<td height="42" style="height:42px;width:183px;">Synthetic Ura- drop out medium</td>
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			<td style="width:99px;">630416</td>
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			<td style="width:99px;">Y20060</td>
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			<td height="42" style="height:42px;width:183px;">Dimethyl Sulfoxide (DMSO)</td>
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			<td style="width:99px;">S67496</td>
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			<td height="42" style="height:42px;width:183px;">Lithium acetate, anhydrous</td>
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			<td style="width:99px;">AC268640010</td>
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			<td height="42" style="height:42px;width:183px;">Polyethylene Glycol 3350 (PEG-3350)</td>
			<td style="width:127px;">Spectrum Chemical&#160;</td>
			<td style="width:99px;">PO125-12KG</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:183px;">96 Pin Replicator&#160;</td>
			<td style="width:127px;">Scinomix</td>
			<td style="width:99px;">SCI-5010-OS</td>
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			<td>140156</td>
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			<td style="width:127px;">Fisher Sci</td>
			<td style="width:99px;">07-200-760</td>
			<td>non-treated, sterile</td>
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			<td style="width:127px;">Eppendorf</td>
			<td style="width:99px;">TI13690052</td>
			<td>30-300ul volume</td>
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			<td height="21" style="height:21px;width:183px;">Eppendorf&#160;MixMate</td>
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			<td height="42" style="height:42px;width:183px;">Eppendorf 5810R Centrifuge</td>
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			<td style="width:99px;">393127</td>
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mating-based overexpression library screening in yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

The ALS-associated protein FUS, an RNA/DNA binding protein, was previously studied in haploid yeast7,8. Genetic screening using the transformation-based method discovered several yeast genes that suppress FUS toxicity. The human homolog of one of the yeast genes was later demonstrated to be effective at suppressing toxicity in a primary neuronal cell and rat model of ALS13. Here, we are using the same yeast...

Watch this Scientific Journal Video about Mating-based Overexpression Library Screening in Yeast at JoVE.com

Mating-based Overexpression Library Screening in Yeast

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

mating-based-overexpression-library-screening-in-yeast

Research

JoVE Journal

Genetics

7.6K Views.  Wright State University. This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

Video: Mating-based Overexpression Library Screening in Yeast

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state when water is supplied. The genomic sequencing of microscopic animals such as tardigrades risks bacterial contamination that sometimes leads to erroneous interpretations, for example, regarding the extent of horizontal gene transfer in these animals. Here, we provide an ultralow input method to sequence the genome of the tardigrade, Hypsibius dujardini, from a single specimen. By employing rigorous washing and contaminant exclusion along with an efficient extraction of the 50 ~ 200 pg genomic DNA from a single individual, we constructed a library sequenced with a DNA sequencing instrument. These libraries were highly reproducible and unbiased, and an informatics analysis of the sequenced reads with other H. dujardini genomes showed a minimal amount of contamination. This method can be applied to unculturable tardigrades that could not be sequenced using previous methods.

Contamination during the genomic sequencing of microscopic organisms remains a large problem. Here, we show a method to sequence the genome of a tardigrade from a single specimen with as little as 50 pg of genomic DNA without whole genome amplification to minimize the risk of contamination.

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

A CRISPR/sgRNA library has been applied to interrogating protein-coding genes.However, the feasibility of a sgRNA library to uncover the function of a CTCF boundary in gene regulation remains unexplored. Here, we describe a HOX loci specific sgRNA library to elucidate the function of CTCF boundaries in HOX loci.

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements ...

Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and lipids during endocytosis, sorting within the endosomal system, and autophagy. Central to SNX-BAR protein function is the ability to form homodimers or heterodimers that bind membranes using highly conserved phox-homology (PX) and BAR (Bin/Amphiphysin/Rvs) domains. In addition, oligomerization of SNX-BAR dimers on membranes can elicit the formation of membrane tubules and vesicles and this activity is thought to reflect their functions as coat proteins for endosome-derived transport carriers. Researchers have long utilized in vitro binding studies using recombinant SNX-BAR proteins on synthetic liposomes or giant unilamellar vesicles (GUVs) to reveal the precise makeup of lipids needed to drive membrane remodeling, thus revealing their mechanism of action. However, due to technical challenges with dual expression systems, toxicity of SNX-BAR protein expression in bacteria, and poor solubility of individual SNX-BAR proteins, most studies to date have examined SNX-BAR homodimers, including non-physiological dimers that form during expression in bacteria. Recently, we have optimized a protocol to overcome the major shortcomings of a typical bacterial expression system. Using this workflow, we demonstrate how to successfully express and purify large amounts of SNX-BAR heterodimers and how to reconstitute them on synthetic liposomes for binding and tubulation assays.

Here, we present a workflow for the expression, purification and liposome binding of SNX-BAR heterodimers in yeast.

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and ...