We thank the Cross lab members for advice and useful discussion. This work was supported by PHS 5RO1-GM078153 and by a Junior Fellow award from the Simons Foundation to Michal Breker.

1. UV Mutagenesis
<ol>
	<li>Prepare a batch of 100 - 200 rectangular agar plates with Tris-Acetate-Phosphate (TAP) medium9,10. Prepare these plates a few days ahead and keep them on the bench to dry to ensure rapid absorption of the suspended cells in the next steps.</li>
	<li>Culture Chlamydomonas cells up to 0.2 - 0.5 optical density (OD750; ~ 2 days) in 100 ml of liquid TAP under light, at 25 &#176;C and shaking at 100 rpm. 
	NOTE: UV mutagenesis is performed independently in two genetic backgrounds: Mat- Hygror (confers resistance to Hygromycin B) and Mat+ Paror (confers resistance to Paromomycin). Antibiotic choice is arbitrary, provided the two mating types have complementary drug resistances.</li>
	<li>Check a sample of each of the cultures under the microscope to ensure that the cells are viable, healthy (swimming and intact), and without contamination. 
	NOTE: Overgrown cultures &#34;crash&#34; and lose viability, appearing as &#34;ghosts&#34; in phase contrast microscopy. Do not keep shaker cultures going once they reach saturation. The &#34;ghost&#34; phenomenon is easily detectable in step 1.3.</li>
	<li>Dilute the culture to 0.003 OD750. Wrap the bottle with aluminum foil to ensure homogenous density, as the strain is motile and swims directionally in response to light.
	<ol>
		<li>Adjust the density of the suspension based on the planned UV dose, so that accounting for cell killing, 200 - 600 survivor colonies will form on the plates (Figure 1). See Table 1 for information on UV exposure times.</li>
	</ol>
	</li>
	<li>Attach a small-tube cassette that fits a liquid dispenser and perform a series of washes for sterilization, according to the manufacturer&#39;s instructions, to prevent contamination.</li>
	<li>Using an 8 x 12 liquid dispenser, dispense 4 x 96 drops of 2 &#181;l each onto rectangular plates (Figure 1). Tap mildly at the edge of the plate to ensure the merging of all drops into a thin sheet of liquid and immediately cover the plates to prevent exposure to light.
	<ol>
		<li>To ensure very even single cell dispersal, use dry plates, as mentioned above, and quickly cover the plates to prevent the cells from swimming in response to light. Keep the covered plates level and in the dark until all liquid is absorbed.</li>
	</ol>
	</li>
	<li>Place the plates under a germicidal UV lamp (30 watt germicidal UV tube) for periods of time determined empirically to give an optimal yield of ts- mutants among the survivors. Here, use times of 0.5 - 1.5 min at a distance of 40 - 50 cm to result in 90 - 99.9% killing. Survivors contain 100 - 1,000 UV-induced point mutations, of which ~ 10% change coding sequences.
	<ol>
		<li>In order to ensure maximum potency of UV irradiation with no reversion due to light-dependent DNA repair11,12, work in the dark at this step and immediately pack the plates in a dark box after irradiation.</li>
	</ol>
	</li>
	<li>Keep the plates in the dark for 8 - 24 hr at room temperature.</li>
	<li>Place the plates in a 21 &#176;C incubator with illumination to form colonies. Colony formation takes ~10 days. Make sure to wrap the plates with plastic bags and add absorbing paper inside to blot up liquid condensation. Evaporation and condensation cycles otherwise can provide liquid film routes for contaminants to enter the plates.</li>
	<li>Load the plates in the relevant stacks as sources for robotic colony picking (Figure 2). Pick colonies for 384-arrays on rectangular plates, and grow them at 21 &#176;C with illumination (~ 1 week).</li>
	<li>Condense the 384-arrays into a 1,536-arrays (4:1) using a replica-plating robot (Figure 3), and allow the plates to grow for ~ 3 days in the 21 &#176;C incubator.</li>
	<li>Replicate the 1,536-arrays to two plates each and place one in the 21 &#176;C incubator (permissive temperature) and the other in a 33 &#176;C incubator (restrictive temperature). Following 24 hr, replicate the plates in 33 &#176;C to a new set of pre-warmed plates, and place them in the 33 &#176;C incubator. 
	NOTE: The reason for this secondary plating is that ts lethal mutants can in some cases accumulate substantial biomass, even if the cells are arrested in the first cell cycle after plating. The secondary replica eliminates this signal and greatly increases sensitivity.</li>
	<li>Photograph the plates with a digital camera following 3 days of growth at 33 &#176;C and 5 days of growth at 21 &#176;C. Hold the plates in a fixed frame. Use a &#34;grid-plate&#34; marked with nine alignment indicators that is photographed together with the culture plates (Figure 4). Photograph culture plates as alternating paired 21 &#176;C and 33 &#176;C (21/33) images. 
	NOTE: Different incubation times are used to equalize growth since the wild type grows significantly faster at 33 &#176;C than at 21 &#176;C.</li>
</ol>
2. Identification of ts Mutant Candidates: First Screen
<ol>
	<li>Process the paired 21/33 plate images with a custom Matlab image analysis software to eliminate the background and to segment the images into a 1,536-array. The program will determine the detected biomass (total pixel intensity) in each position (Figure 5). 
	NOTE: The software (provided in the S.I. along with instructions) will use the grid-plate image to determine the locations of cells (individual entries) in a 1,536-array at the magnification and plate alignment used and calculate total pixel intensity in each cell. These values for each mutant are then compared against adjustable parameters to determine required growth at 21 &#176;C relative to a ts+ standard and the degree of temperature-sensitivity, defined as: S(Mut33)/S(WT33)/S(Mut21)/S(WT21), where S is signal (pixel intensity after background subtraction), Mut is an individual mutant, and &#34;WT&#34; is a randomly chosen non-temperature-sensitive colony (mutagenized strain that had ts+ phenotype). Growth at 21 &#176;C is defined as S(Mut21)/S(WT21). In this first screen, apply relaxed selection criteria (allowing relatively low growth at 21 &#176;C and a relatively low degree of temperature sensitivity) to keep the false negative rate low.</li>
	<li>Load the list of selected colonies generated by the software as an instruction file for the single-colony picking robotics (typically into a 384-array). Prepare the source and target plates according to the robotics instructions and pick colonies to array (Figure 6). 
	NOTE: Conventional pickers require an instruction file in some format, such as .csv, .txt, or .xls. All pickers will have the capacity to be file-driven; different formats will require minor editing of the MATLAB code (source code provided).</li>
	<li>Place the target plates in the 21 &#176;C incubator for ~ 5 days to grow a stock plate.</li>
</ol>
3. Identifying ts Mutants: Second Screen
<ol>
	<li>Repeat step 2.1 to retest picked colonies. Typically 30 - 50% of colonies will retest as clear ts lethals, for a yield of 2% - 5% ts lethals relative to initial colonies surviving UV mutagenesis.</li>
	<li>Repeat step 2.2 for picking the twice-screened ts lethals, but with a modified instruction file designed to array colonies into blocks of 100 colonies on rectangular plates at a 384-density. This is for convenient microscopic examination in the next step. The instruction file format is specified at runtime by the MATLAB code.</li>
	<li>Make sure to include several WT colonies as a control and place the plates at 21 &#176;C for ~ 5 days.</li>
</ol>
4. Initial Phenotype Determination
<ol>
	<li>Replicate the 100-block plates arrayed in step 3.2 and place them in the 21 &#176;C incubator for ~ 2 days to obtain freshly growing colonies.</li>
	<li>Replicate the fresh copy of the 100-block plates (4.1) to three copies. Place one copy in the 21 &#176;C incubator and one in the 33 &#176;C incubator as controls.</li>
	<li>Place the third copy (&#34;screening plates&#34;) in a robotic setup, and spot the colonies with long pins touched with sterilized water. 
	NOTE: Chlamydomonas cells pinned to agar robotically tend to land initially as a rather dense spot of cells, with few single cells available for microscopic inspection. To optimize the colonies for microscopic inspection, spot the initially transferred cells with a drop of water (the volume of water transferred by the robotic long pins is ~ 100 nl). This results in dispersion of the cells in a small radius (~ 1 mm) about the initial pinning center, with many isolated single cells.</li>
	<li>Take photomicrographs of a region of each spot of the screening plates at time 0 (while still single, undivided cells) (Figure 7) and place the plates at 33 &#176;C for incubation. Determine image location by the manual stage control of a modified tetrad dissection microscope set to give stops at the 4.5-mm center-to-center spacing of a 384-density array.</li>
	<li>Remove the screening plates from the 33 &#176;C incubator and take photomicrographs, as in step 4.4, at varying time points (10 hr, 20 hr, 48 hr). Make sure the stage, the plate holder, and the stage controller are precisely calibrated to get images of the same cells in every time point. Perform quick photomicrograph acquisition (~ 10 min).</li>
	<li>Use the second copy in the 33 &#176;C incubator to verify the ts phenotype and to make sure that temperature fluctuations during image acquisition have no major effect on ts phenotype.
	<ol>
		<li>Analyze microscopic images and select for mutants based on the desired criteria (Figure 8). Spot the final selected set in a 96-arrayed agar plate. Make sure that each plate contains mutants of the same mating type and drug resistance.</li>
		<li>For each plate, incorporate the last two columns a positive (query mutation) and a negative (WT) control for the complementation assay.</li>
	</ol>
	</li>
</ol>
5. Complementation and Linkage Testing of New Mutants to &#34;Frequent Flyers&#34;
<ol>
	<li>Transfer large amounts of the arrayed colonies to nitrogen-free gamete-induction medium10 in 96-well microplates (density of each colony should be around 0.2 O.D.). Incubate the plates under light (13 - 20 W mercury bulbs) for ~ 5 hr to allow gametogenesis. 
	NOTE: This step can be conducted either with a replicating robotic setup or manually with a simple plating device.</li>
	<li>Suspend queries with the opposite mating types harboring the alternative resistance cassettes into tubes with nitrogen-free gamete-induction medium10 for gametogenesis.</li>
	<li>Mix the samples in a target plate in a mating mixture volume of 20 &#181;l (Figure 9). Following ~10 min under the light, spot 5 &#181;l from each well twice: once on a TAP plate for linkage testing and once on TAP + 5 &#181;M Paro + 9 &#181;M Hygro for complementation testing.</li>
	<li>Incubate the linkage plates in the 21 &#176;C incubator overnight, and then wrap them in foil. Keep them in the dark for 5 days to allow zygospore formation.</li>
	<li>Incubate complementation-testing plates for ~10 days in the light at 21 &#176;C. 
	NOTE: The drug amounts are calibrated to allow survival of doubly heterozygous diploids, but they are still enough to eliminate the mating haploids. These amounts were calibrated for standard resistance cassette integrations used in this specific project; with new integrations, doses should be recalibrated. Most biomass is verified to be diploids by flow cytometry (Figure 9), although a variable level of haploids (probably from meiosis and growth of doubly resistant segregants) is usually observed as well.</li>
	<li>Replicate the complementation-testing plates into two copies for ts- phenotype identification. Place one copy at 21 &#176;C and one copy at 33 &#176;C for ~ 5 days.</li>
	<li>Test colonies for the ts- phenotype, as described in section 2.1 (Figure 5). Colonies that are not complementing with the query likely represent alleles of the same gene as the query (diploids hetero-allelic for mutations in the same gene).</li>
	<li>For linkage testing, following step 5.4, shift the plates back to light to allow meiosis and outgrowth of haploid segregants for ~7 days.</li>
	<li>Replicate linkage testing plates to TAP + 10 &#181;M Paro and 10 &#181;M Hygro to select for double-resistant progenies (predicted to be 25% of the haploid progeny, since the cassettes are unlinked) and incubate at 21 &#176;C for a week. 
	NOTE: Increase the drug dosage for doubly resistant haploids.</li>
	<li>Test colonies for the ts- phenotype, as in step 2.1. 
	NOTE: A ts- phenotype is expected (and observed) for mutants in the same complementation group as the query. In addition, a ts- phenotype in the linkage assay, for a mutant that complements the query, can reflect tight linkage between the query and the new mutation. Mutants that complement the tested queries are likely new genes that will enter the pipeline for bioinformatic identification of the causative lesion, and ultimately for further phenotypic analysis.</li>
</ol>

<ol>
	<li>Tulin, F., Cross, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microbial+avenue+to+cell+cycle+control+in+the+plant+superkingdom.">A microbial avenue to cell cycle control in the plant superkingdom.</a> Plant Cell. 26, 4019-4038 (2014).</li><li>Cross, F. R., Umen, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Chlamydomonas+cell+cycle.">The Chlamydomonas cell cycle.</a> Plant J. 82, 370-392 (2015).</li><li>Li, X., 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=An+Indexed,+Mapped+Mutant+Library+Enables+Reverse+Genetics+Studies+of+Biological+Processes+in+Chlamydomonas+reinhardtii.">An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii.</a> Plant Cell. 28, 367-387 (2016).</li><li>Tulin, F., Cross, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin-Dependent+Kinase+Regulation+of+Diurnal+Transcription+in+Chlamydomonas.">Cyclin-Dependent Kinase Regulation of Diurnal Transcription in Chlamydomonas.</a> Plant Cell. 27, 2727-2742 (2015).</li><li>Simchen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+mutants.">Cell cycle mutants.</a> Annu. Rev. Genet. 12, 161-191 (1978).</li><li>Hartwell, L. H., Culotti, J., Reid, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+control+of+the+cell-division+cycle+in+yeast.">Genetic control of the cell-division cycle in yeast.</a> I. Detection of mutants. Proc. Natl. Acad. Sci. U. S. A. 66, 352-359 (1970).</li><li>Li, 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=Systematic+exploration+of+essential+yeast+gene+function+with+temperature-sensitive+mutants.">Systematic exploration of essential yeast gene function with temperature-sensitive mutants.</a> Nat. Biotechnol. 29, 361-367 (2011).</li><li>Ben-Aroya, 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=Toward+a+comprehensive+temperature-sensitive+mutant+repository+of+the+essential+genes+of+Saccharomyces+cerevisiae.">Toward a comprehensive temperature-sensitive mutant repository of the essential genes of Saccharomyces cerevisiae.</a> Mol Cell. 30, 248-258 (2008).</li><li>Harris, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Chlamydomonas Sourcebook: Introduction into Chlamydomonas and Its Laboratory Use. , (2008).</li><li>Dutcher, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mating+and+tetrad+analysis+in+Chlamydomonas+reinhardtii.">Mating and tetrad analysis in Chlamydomonas reinhardtii.</a> Methods Cell Biol. 47, 531-540 (1995).</li><li>Gill, S. S., Anjum, N. A., Gill, R., Jha, M., Tuteja, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+and+repair+in+plants+under+ultraviolet+and+ionizing+radiations.">DNA damage and repair in plants under ultraviolet and ionizing radiations.</a> Sci World J. 2015, 250158 (2015).</li><li>Liu, Z., Wang, L., Zhong, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+and+mechanisms+of+DNA+repair+by+photolyase.">Dynamics and mechanisms of DNA repair by photolyase.</a> Phys. Chem. Chem. Phys. 17, 11933-11949 (2015).</li><li>Francis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+control+and+plant+development.+Annual+Plant+Reviews,+Volume+32.">Cell cycle control and plant development. Annual Plant Reviews, Volume 32.</a> Ann. Bot. 101, 1049-1050 (2008).</li><li>Zachariae, W., Nasmyth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whose+end+is+destruction:+cell+division+and+the+anaphase-promoting+complex.">Whose end is destruction: cell division and the anaphase-promoting complex.</a> Genes Dev. 13, 2039-2058 (1999).</li><li>Merchant, S. 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=The+Chlamydomonas+genome+reveals+the+evolution+of+key+animal+and+plant+functions.">The Chlamydomonas genome reveals the evolution of key animal and plant functions.</a> Science. 318, 245-250 (2007).</li><li>Ben-Aroya, 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=Toward+a+comprehensive+temperature-sensitive+mutant+repository+of+the+essential+genes+of+Saccharomyces+cerevisiae.">Toward a comprehensive temperature-sensitive mutant repository of the essential genes of Saccharomyces cerevisiae.</a> Mol. Cell. 30, 248-258 (2008).</li><li>Mnaimneh, 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=Exploration+of+essential+gene+functions+via+titratable+promoter+alleles.">Exploration of essential gene functions via titratable promoter alleles.</a> Cell. 118, 31-44 (2004).</li><li>Schuldiner, 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=Exploration+of+the+function+and+organization+of+the+yeast+early+secretory+pathway+through+an+epistatic+miniarray+profile.">Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile.</a> Cell. 123, 507-519 (2005).</li><li>Hsu, P. D., 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=DNA+targeting+specificity+of+RNA-guided+Cas9+nucleases.">DNA targeting specificity of RNA-guided Cas9 nucleases.</a> Nat. Biotechnol. 31, 827-832 (2013).</li><li>Jiang, W., Brueggeman, A. J., Horken, K. M., Plucinak, T. M., Weeks, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+transient+expression+of+Cas9+and+single+guide+RNA+genes+in+Chlamydomonas+reinhardtii.">Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii.</a> Eukaryot. Cell. 13, 1465-1469 (2014).</li></ol>

The authors declare no significant financial interests.

The pipeline described here for high yield isolation of ts lethal mutants ensures that presumably all cellular-essential pathways of the Chlamydomonas genome are represented. The two most critical steps for efficient collection of potential cell cycle genes and for the elimination of repetitive &#34;frequent flyer&#34; alleles are: 1) the coherent definition of arrest phenotype characteristics for incomplete cell cycles and 2) the parallel complementation assay against already-identified query genes to enlarge the collection with newly isolated ones.
When synchronized by light-dark cycles, Chlamydomonas grows photosynthetically during daylight hours and increases in cell size &#62; 10x without any DNA replication or cell division13. Approximately coincident with the onset of night, cells then undergo multiple cycles of alternating DNA replication, mitosis, and cell division (Figure 8). This regulatory scheme provides a natural distinction between genes primarily required for cell growth and integrity and genes required specifically for the cell division cycle. We found that the 10-hr and 20-hr time points are very informative for an initial rough phenotypic cut1. The broad classes of ts lethal mutants that we recognize currently, based on these images (see S.I. in Tulin and Cross, 2014)1, are: Notch, Popcorn, Round, Small, Medium, early lysis, and multiple-cycle (Figure 8).
The three most relevant categories we focus on are Notch, Popcorn, and Round. The &#34;Notch&#34; and &#34;Popcorn&#34; phenotypes were shown previously to be characteristic of most cell cycle-specific lesions (e.g., mitotic cyclin-dependent kinase, DNA replication machinery, and Topoisomerase II)1. The appearance of one (Notch) or multiple (Popcorn) apparent planes of incipient but unsuccessful cell division is a convenient morphological indicator of cell cycle initiation. These mutants generally exhibit little or no growth defects, with increases in cell volume similar to WT at the 10-hr mark. The Notch and Popcorn phenotypes are evident at 10 hr and are fully developed (frequently associated with cell lysis) by 20 hr. &#34;Round&#34; cells grow similarly to WT but with much-reduced production of apparent incipient division planes, thus yielding large, round arrested cells. Previous mutants in this category have fallen into components of the anaphase-promoting complex14 or in genes required for microtubule function (tubulin-folding cofactors, gamma-tubulin ring complex)1. At later times, these cells frequently exhibit pronounced cell lysis.
&#34;Small&#34; and &#34;Medium&#34; cells grow either negligibly (Small) or significantly less than WT (Medium). Many of these mutants identified to date have lesions in genes whose annotations suggest roles in basic cellular growth processes (translation or membrane biogenesis). The main microscopic discrimination between Medium and Round rests on the amount of growth at 10 hr (Round: like WT; Medium: reduced). Because the Small and Medium categories are quite large and probably reflect lesions in a great range of cellular pathways, we are not attempting to saturate these categories; however, we do want to molecularly identify representatives of the class to understand phenotypes of loss in diverse pathways. Two unstudied categories are: 1) the early-lysing mutants that lose integrity (loss of green color, loss of refractility) by the 10-hr mark, with little evidence of prior cell growth and 2) the multiple cycles. Cells proliferate similarly to WT at 10 and 20 hr, though they exhibit a complete inability to carry out longer-term proliferation.
We are mostly characterizing &#34;notch,&#34; &#34;popcorn,&#34; and &#34;round&#34; and exclude small and medium round cells, as well as leaky mutants that complete few cell divisions. This is principally to ensure that basic cellular features, such as growth and membrane integrity, are functional and enrich the probability for division-related genes. This approach is found to be empirically efficient; however, it may be that a cell cycle gene is pleiotropic and has additional roles earlier in G1, before actual division. Such cases, which we expect to be rare, are missed. More generally, we aim for homogenous arrest, which in high probability is due to one causative mutation that is a completely dysfunctional protein. However, for the same reason as just described, there may be several arrest points and therefore, some flexibility is advisable in choosing candidates.
In order to enrich the collection with newly identified genes, the chosen candidates are assayed for complementation. We require ts- in the positive control (query against query mutation) and ts+ in the negative control (query against WT). New mutants in the same complementation group as the query are ts-. Membership in the same complementation group almost invariably reflects a molecular lesion in the same gene (this has been the case for every such gene we have tested). Therefore, for &#34;frequent flyers,&#34; this criterion is exclusionary for further characterization. Mutants that were not at the same complementation groups as the tested queries are candidates for new genes and are further characterized by bioinformatics and experimental tools. Highly variable recovery of ts- alleles into different complementation groups is a well-known phenomenon-that is, variability is markedly greater than Poisson noise, due to the great intrinsic variability of mutability to ts- between different genes. Causes could include intrinsic thermolability differences; different protein sizes; the presence of a protein as a monomer versus as a large, stabilized complex; and mutagenic hot spots. This is almost a pure nuisance. However, one resulting favorable outcome is that the &#34;frequent flyer&#34; list is not long (with only a few targets occupying most of the list), so the labor-intensive complementation testing is not a massive undertaking until the later stages of the project.
As a complementary approach, we performed a linkage assay. In this assay, double-resistant progenies are selected and are tested for the ts- phenotype. A ts- phenotype is expected (and observed) for mutants in the same complementation group as the query or for tightly linked mutations. For each of the tested genes, a WT progeny is expected to appear (ts+ phenotype) in a certain probability depending on the genetic distance. We estimate that there are around 100 zygospores for each mating in these spots. Assuming 100% meiotic efficiency, this will result in around 100 double-drug-resistant progeny from the unlinked drug resistance cassettes (25% of the meiotic progeny, four per meiosis, due to Mendelian inheritance). This would also be the case for ts- mutations, where 25% of the progenies will be double-mutant, and 25% will be WT if the query and test mutations are unlinked. Therefore, out of the double-drug-resistant progeny, 25% will be WT (around 25 cells). This is the case for completely unlinked mutations; however, moderate linkage (within ~ 20 cM, about 2 Mb, or 2% of the genome115) will strongly reduce or eliminate the ts+ signal. In the case of linkage of the tested mutation to the antibiotic cassette, ts+ haploids that are double-drug-resistant are present in very low amounts. This manifests as apparent failure to recombine with all mutants tested, despite complementing all mutants tested, an aberrant result that is easily noted; in such cases, backcrossing will solve the problem.
Both from prior knowledge and from sequence analysis, we expect cell cycle genes in the Chlamydomonas to be around 500 genes2, although most, but probably not all, are essential. We will evaluate the necessity for additional mutagenesis rounds as more mutants are collected and the level of saturation rises.
This procedure is uniquely designed for studying essential biological processes and the genes and proteins that carry them out. Other methodologies to generate perturbations in essential genes exist (e.g., transformation of randomly mutagenized alleles16, conditionally transcribed alleles17, or hypomorphic alleles18). However, they all require homologous recombination, which is strongly suppressed in vegetative Chlamydomonas. The clustered, regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been established as a powerful tool for gene modification19; however, it is yet to work efficiently in Chlamydomonas20. Critically, all of these methods require prior knowledge of the target. This is a severe restriction if one wishes to have the possibility to learn something new! Our approach will yield mutations identifying essential genes, independent of any prior knowledge. Therefore, at the present level of technology, isolation of random ts mutations followed by gene identification by deep sequencing may be the most efficient method of gaining rapid entry into microbial cell biology in the plant superkingdom.
Identification of causative mutations (from among ~100 coding-sequence-changing mutations in each clone) is beyond the scope of this paper. Deep sequencing of bulked segregant pools1 is effective but labor-intensive. A combinatorial pool strategy for the determination of all mutations in a large number of strains, after sequencing a small number of pools, is very cost- and labor-effective. A new strategy for combinatorial bulked segregant sequencing is under development that will allow the identification of causative mutations in dozens of mutants simultaneously in a single sequencing run (in preparation). These efficiencies are very important to allow the critical gene identification step to keep pace with the very rapid accumulation of mutants that is made possible by the procedures described here.

Phenotypic characterization of systematic mutant collections of model organisms is a proven approach for dissecting cellular complexity. The haploid unicellular green algae Chlamydomonas reinhardtii has a plant-like gene set, but it diverged from land plants before multiple genome duplications in the land plant lineage2. In principle, lack of gene duplication and a mainly haploid life cycle greatly facilitates loss-of-function genetic approaches. However, targeted disruption of genes of interest is nearly impossible due to the lack of efficient homologous genomic integration. A random insertional disruption library is under construction, combined with identification of the disrupted site, so far yielding an arrayed set of 1,935 mapped disruptions representing 1,562 genes3. However, this approach (expected in general to produce null mutations) is not applicable to essential genes. Temperature-sensitive (ts) mutations can be recovered in essential genes, and recent methods allow efficient identification of the mutated gene and the causative lesion. Phenotypic analysis at high temperature then provides immediate information about the function of the mutated gene. We reported on the isolation and characterization of ts lethal mutations in ~70 essential genes in Chlamydomonas, focusing especially on genes involved in cell cycle progression and control1,4.
Ts lethal screens have been a mainstay of genetic analysis in microorganisms for decades5,6. In principle, a desirable feature is to approach &#34;saturation,&#34; meaning that all genes capable of mutating to ts lethality are identified by at least one mutant, allowing a complete analysis. However, in practice, several factors limit the approach to saturation. First, while almost all genes can be mutated to loss of activity at high temperature, the efficiency of recovery of such mutants varies over at least an order of magnitude7,8. Therefore, a random screen begins to pick up recurrent hits in &#34;frequent flyers&#34; long before saturation is approached. Second, while ts mutations usually result in reduction of function, they may not be true nulls at a restrictive temperature (and conversely, are frequently not fully functional at a permissive temperature). This problem can be dealt with to some extent by comparing multiple alleles; if they all share a common phenotype, this is more likely to reflect the result of simple inactivation of the gene. Multiple alleles are also very helpful for definitive molecular identification of the causative lesion1. However, the &#34;frequent flyer&#34; problem means that multiple alleles in rarely hit genes can be difficult to recover.
For these reasons, we have been developing an enhanced pipeline to isolate and phenotypically characterize ts mutants. We have collected over 3,000 ts mutants so far, including about 200 new candidate cell cycle mutations. Molecular and phenotypic analysis of this collection, which already likely includes mutations in most or all cell-essential pathways, should provide new insights and hypotheses in plant cell biology. Importantly, this pipeline can be applied to any microorganism that grows on agar to efficiently construct ts mutant collections.
A note on equipment: two robots are very important for efficiency in this procedure (a colony picker and a combination replica plater/cherry picker). Pickers typically have metal pins. Picked colonies are held in air on these pins for some period (seconds to minutes, depending on the model). Chlamydomonas dies in about 20 sec on a metal pin in air. This restricts model choice for the organism. Accuracy matters. Our colony picker is reasonably accurate; however, it is somewhat violent in its action and has some possibility for spray-based cross-contamination. It is not used at higher than the 384 density (4.5 mm center-center spacing); at this density, the accuracy is completely acceptable. The replica plating/cherry picking robot (different attachments used for these applications) is much slower at picking, but is accurate enough for the 1,536 density (6,144 is a challenge, even for this very accurate robot; we have evaluated this density but elected not to use it because of its various difficulties). The robot will not work well if plates are loaded unevenly, etc. It is important to spot-check to be sure the right things are happening; of course, the robot will run unattended and in general, all will go well if the first few plates were correct.

<table border="0" cellpadding="0" cellspacing="0" width="672">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:293px;">Equipment:</td>
			<td style="width:144px;"></td>
			<td style="width:147px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hudson RapidPick colony picker</td>
			<td>Hudson Robotics</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MultiDrop Combi Reagent Dispenser</td>
			<td>Thermo Scientific</td>
			<td>5840300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small tube metal tip cassette</td>
			<td>Thermo Scientific</td>
			<td>24073295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Singer RotoR replica-plating robot</td>
			<td>Singer Instruments</td>
			<td></td>
			<td>Very essential for the process. For lower scale screenings you can use an in-house manual tool</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Singer single-colony picking attachment (&#8216;Stinger&#8217;)</td>
			<td>Singer Instruments</td>
			<td></td>
			<td>Can be picked manually, however for large scales it is nearly impossible</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYTOX Green Nucleic Acid Stain</td>
			<td>ThermoFisher Scientific</td>
			<td>S7020</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput robotically assisted isolation of temperature-sensitive lethal mutants in chlamydomonas reinhardtii

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the conventional approaches of permanent gene deletion cannot be applied to essential genes. We have pioneered a unique collection of ~70 temperature-sensitive (ts) lethal mutants for studying cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii1. These mutations identify essential genes, and the ts alleles can be conditionally inactivated by temperature shift, providing valuable tools to identify and analyze essential functions. Mutant collections are much more valuable if they are close to comprehensive, since scattershot collections can miss important components. However, this requires the efficient collection of a large number of mutants, especially in a wide-target screen. Here, we describe a robotics-based pipeline for generating ts lethal mutants and analyzing their phenotype in Chlamydomonas. This technique can be applied to any microorganism that grows on agar. We have collected over 3000 ts mutants, probably including mutations in most or all cell-essential pathways, including about 200 new candidate cell cycle mutations. Subsequent molecular and cellular characterization of these mutants should provide new insights in plant cell biology; a comprehensive mutant collection is an essential prerequisite to ensure coverage of a broad range of biological pathways. These methods are integrated with downstream genetics and bioinformatics procedures for efficient mapping and identification of the causative mutations that are beyond the scope of this manuscript.

We show an accelerated pipeline for isolating ts mutants in Chlamydomonas. Cells are dispensed on agar plates, and shortly after quick validation under the microscope for single-cell density, plates are UV irradiated (Figure 1). Typical irradiated colonies are identified and picked into an arrayed format after 10 days of growth at a permissive temperature (Figure 2). The resultant plates in the 384 format are merged to a 1,536-array (Figure 3). From these first steps of collecting irradiated colonies, we have arrayed ~ 200,000 colonies so far. The density of the suspension is adjusted based on the planned UV dose so that, accounting for death, 200 - 600 survivor colonies will form on the plates. Three UV exposure times (1.5 min, 1 min, and 0.5 min) and three densities were tested accordingly (Table 1). Empirically, the 1.5 min exposure time yielded most of the ts- mutants (~ 50%); however, by far, 1 min yielded most of the cell cycle candidates. Therefore, future UV mutagenesis rounds were done with 1 min only. An important concern is the splashing during initial picking and the cross contamination (especially in the high-density formats). This may result in duplication and further phenotyping of the same mutant twice. To minimize the probability for such a scenario, take care to collect colonies at the optimal size, and make sure that adjacent colonies are not picked in the initial assay.
Next, two sequential ts- phenotype assays (Figure 5) were performed, and around 3,000 ts- mutants were isolated and phenotypically characterized by time-lapse microscopy (Figure 8). Due to the biological focus in studying the cell cycle of interest in phenotypes that meet certain criteria, we are not interested in collecting more than two alleles in each target gene. Therefore, we performed complementation and linkage testing with already characterized genes with more than two alleles (Query) against newly collected candidates to remove highly recurrent genes from the downstream pipeline (Figure 9).
<img alt="Figure 1" src="/files/ftp_upload/54831/54831fig1.jpg" /> 
Figure 1: Uniform Spreading of Unmutagenized Cells and UV Mutagenesis. (a) 384 x 2 &#181;l drops are dispensed on an agar plate using a reagent dispenser. (b) Random plates are tested under the microscope to verify single-cell density and are UV irradiated with a 1-min exposure. Scale bar = 50 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/54831/54831fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54831/54831fig2.jpg" /> 
Figure 2. UV-mutagenized Colonies Picked for the 384-arrays. (a) UV-irradiated single cells are grown for ~10 days into visible colonies. Scale bar = 2 cm. (b) Image analysis program recognizes separable colonies according to certain parameters and robotically arrays them into 384 plates. <a href="http://cloudflare.jove.com/files/ftp_upload/54831/54831fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54831/54831fig3.jpg" /> 
Figure 3. Merging to the 1,536-array. Four 384-format plates are merged to one 1,536-plate; once grown, they are replicated again to test for the ts- phenotype. <a href="http://cloudflare.jove.com/files/ftp_upload/54831/54831fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54831/54831fig4.jpg" /> 
Figure 4. Imaging for Quantification. (a) Plates are held in a frame under a document photography stand so that all plates in a series are photographed at identical magnification and positioning. (b) The first image is of a 1536-well microtiter plate with red dots placed at corners and midpoints. This &#34;grid-plate&#34; image defines the position of the array. (c) Example of a 1,536-array after incubation. <a href="http://cloudflare.jove.com/files/ftp_upload/54831/54831fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54831/54831fig5.jpg" /> 
Figure 5. Identification of the ts- phenotype by Semi-automated Image Analysis. Plate images are analyzed by custom MATLAB software (provided in the S.I.). The image is automatically segmented into cell arrays (96, 384, or 1,536), and the signal in each cell is quantified. Growth at 21 &#176;C is marked in blue, and growth at 33 &#176;C is marked in yellow. Colonies exhibiting significantly higher growth at 21 &#176;C compared to 33 &#176;C (standardized to ts+) are selected and marked in black squares with a green dot. These choices can be edited manually. Final selections are transferred to a file used by the colony-picking robot. <a href="http://cloudflare.jove.com/files/ftp_upload/54831/54831fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54831/54831fig6.jpg" /> 
Figure 6. Robotic Picking of First-round ts Lethal Candidates. The cherry-picking robot follows instructions from a file generated as described in step 2.1 to pick candidates for a new array. The top panel shows the loading of a sterilized pin. The bottom panel shows the precise picking from a certain colony in the source plate. <a href="http://cloudflare.jove.com/files/ftp_upload/54831/54831fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54831/54831fig7.jpg" /> 
Figure 7. Time-lapse Screening for Initial Sorting of ts Lethal Phenotypes. Clones that passed two rounds of the screening protocol illustrated in Figure 5 are replicated to plates at a cell density such that individual cells can be resolved microscopically. A modified tetrad dissection microscope is used to identify positions at which photomicrographs are taken after 0 hr, 10 hr, and 20 hr incubations at 33 &#176;C. <a href="http://cloudflare.jove.com/files/ftp_upload/54831/54831fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/54831/54831fig8.jpg" /> 
Figure 8: The Chlamydomonas Cell Cycle Mutants Identified by Time-Lapse Microscopy. (a) Illustration of the Chlamydomonas unique cell cycle describes the long G1 phase characterized by cellular growth, followed by fission S-M cycles, which end up with the hatching of newborn daughter cells. (b) Microscopic images demonstrate WT cells during the cell cycle and the identification of typical cell cycle mutants that fail to complete mitosis. Scale bar = 50 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/54831/54831fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" src="/files/ftp_upload/54831/54831fig9.jpg" /> 
Figure 9: Complementation and Linkage Test. (a) An antibiotic resistant (e.g., Hygro) query with a frequently repeated mutated gene is crossed in a 96-well plate with a set of mutants of the opposite mating type that harbor a second antibiotic resistance cassette (e.g., Paro). Complementation plates yield high fraction of diploids as shown by the nucleic acid staining and analysis in flow cytometry (lower left panel in a). (b) The mating mixture is tested both for complementation in diploids and for linkage in double-resistant meiotic progeny. The positive control is the query itself in the opposite mating type, and, as expected, shows the ts- phenotype at 33 &#176;C, whereas the negative control is WT and shows the ts+ phenotype. The circled colonies show no complementation with the query and are therefore new ts- alleles for the query gene. These are generally excluded from further characterization, since only &#34;frequent flyers&#34; are in the complementation test set. <a href="http://cloudflare.jove.com/files/ftp_upload/54831/54831fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time</td>
			<td>O.D.</td>
			<td>Colonies picked (#)</td>
			<td>Ts- phenotype</td>
			<td>Cell-cycle candidates</td>
		</tr>
		<tr>
			<td>1.5&#8217;</td>
			<td>0.012</td>
			<td>6000 (Avg=~200)</td>
			<td>200 (3.3%)</td>
			<td>7 (3.5%)</td>
		</tr>
		<tr>
			<td rowspan="2">1&#8217;</td>
			<td rowspan="2">0.003</td>
			<td>11,000</td>
			<td rowspan="2">131 (1.19%)</td>
			<td rowspan="2">15 (11.4%)</td>
		</tr>
		<tr>
			<td>(Avg=~360)</td>
		</tr>
		<tr>
			<td rowspan="2">0.5&#8217;</td>
			<td rowspan="2">6E-04</td>
			<td>9000</td>
			<td rowspan="2">40 (0.45%)</td>
			<td rowspan="2">1 (2.5%)</td>
		</tr>
		<tr>
			<td>(Avg=~300)</td>
		</tr>
	</tbody>
</table>
Table 1: Calibration of Irradiation Times to Maximize the Yield of Cell Cycle Mutant Candidates. Three irradiation times were tested (30 plates each) with corresponding ODs to ensure surviving colony numbers (200 - 600).

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High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii | Genetics | Video

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Temperature-sensitive (ts) lethal mutants are valuable tools to identify and analyze essential functions. Here we describe methods to generate and classify ts lethal mutants in high throughput.

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the ...

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9.7K Views.  The Rockefeller University. Temperature-sensitive (ts) lethal mutants are valuable tools to identify and analyze essential functions. Here we describe methods to generate and classify ts lethal mutants in high throughput. 

Video: High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

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 ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...