Name: high-throughput dna plasmid multiplexing and transfection using acoustic nanodispensing technology
Uploaded: 2019-08-08T13:00:00
Description: Watch this Scientific Journal Video about High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology at JoVE.com

The authors disclosed a receipt of the following financial support for the research, authorship, and/or publication of this article: Inserm, Lille University, Lille Pasteur Institute, Conseil R&#233;gional du Nord, and PRIM-HCV1 and 2 (P&#244;le de Recherche Interdisciplinaire sur le M&#233;dicament), Agence Nationale de la Recherche (ANR-10-EQPX-04-01), the Feder (12001407 (D-AL) Equipex Imaginex BioMed) and the European Community (ERC-STG INTRACELLTB n&#176; 260901). The authors wish to thank Dr. S. Moureu, Dr. B. Villemagne, Dr. R. Ferru-Cl&#233;ment, and Dr. H. Groult for their critical review and corrections of the manuscript.

1. Advance preparations
<ol>
	<li>Preparation of the peristaltic liquid handler programs 
	NOTE: For the diluent and cell dispensing steps of the protocol, a dedicated program must be prepared, taking into account the height of the dispensing head to the plate used and the step intent.
	<ol>
		<li>For the 1 &#181;L diluent dispense step, mount a 1 &#181;L cassette and prepare a program with the settings as described in steps 1.1.1.1 and 1.1.1.2.
		<ol>
			<li>Adjust the flow rate parameter to <str...

<ol>
	<li>Colin, B., Deprez, B., Couturier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+DNA+Plasmid+Transfection+Using+Acoustic+Droplet+Ejection+Technology.">High-Throughput DNA Plasmid Transfection Using Acoustic Droplet Ejection Technology.</a> SLAS Discovery: Advancing Life Sciences R &#38; D. , 2472555218803064 (2018).</li><li>Mirus Bio. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optimising Transfection Performance. , (2019).</li><li>Thermo Fisher Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Factors Influencing Transfection Efficiency | Thermo Fisher Scientific - FR. , (2019).</li><li>Boussif, O., 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+versatile+vector+for+gene+and+oligonucleotide+transfer+into+cells+in+culture+and+in+vivo:+polyethylenimine.">A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.</a> Proceedings of the National Academy of Sciences of the United States of America. 92 (16), 7297-7301 (1995).</li><li>Figueroa, E., 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+mechanistic+investigation+exploring+the+differential+transfection+efficiencies+between+the+easy-to-transfect+SK-BR3+and+difficult-to-transfect+CT26+cell+lines.">A mechanistic investigation exploring the differential transfection efficiencies between the easy-to-transfect SK-BR3 and difficult-to-transfect CT26 cell lines.</a> Journal of Nanobiotechnology. 15 (1), 36 (2017).</li><li>Kirchenbuechler, I., Kirchenbuechler, D., Elbaum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+cationic+lipid-based+transfection+and+cell+division.">Correlation between cationic lipid-based transfection and cell division.</a> Experimental Cell Research. 345 (1), 1-5 (2016).</li><li>Zhang, Z., Qiu, S., Zhang, X., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+DNA+electroporation+for+primary+human+T+cell+engineering.">Optimized DNA electroporation for primary human T cell engineering.</a> BMC Biotechnology. 18 (1), 4 (2018).</li><li>Cao, 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=Transfection+activity+and+the+mechanism+of+pDNA-complexes+based+on+the+hybrid+of+low-generation+PAMAM+and+branched+PEI-1.8k.">Transfection activity and the mechanism of pDNA-complexes based on the hybrid of low-generation PAMAM and branched PEI-1.8k.</a> Molecular bioSystems. 9 (12), 3175-3186 (2013).</li><li>Bos, A. B., 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=Development+of+a+semi-automated+high+throughput+transient+transfection+system.">Development of a semi-automated high throughput transient transfection system.</a> Journal of Biotechnology. 180, 10-16 (2014).</li><li>Colosimo, 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=Transfer+and+expression+of+foreign+genes+in+mammalian+cells.">Transfer and expression of foreign genes in mammalian cells.</a> BioTechniques. 29 (2), 314-318 (2000).</li><li>Villa-Diaz, L. G., Garcia-Perez, J. L., Krebsbach, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+transfection+efficiency+of+human+embryonic+stem+cells+by+the+incorporation+of+DNA+liposomes+in+extracellular+matrix.">Enhanced transfection efficiency of human embryonic stem cells by the incorporation of DNA liposomes in extracellular matrix.</a> Stem Cells and Development. 19 (12), 1949-1957 (2010).</li><li>Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Reverse transfection method. , WO2001020015A1 (2001).</li><li>Raymond, C., 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+simplified+polyethylenimine-mediated+transfection+process+for+large-scale+and+high-throughput+applications.">A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications.</a> Methods (San Diego, CA). 55 (1), 44-51 (2011).</li><li>Junquera, E., Aicart, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+gene+therapy+to+deliver+nucleic+acids+with+multivalent+cationic+vectors.">Recent progress in gene therapy to deliver nucleic acids with multivalent cationic vectors.</a> Advances in Colloid and Interface Science. 233, 161-175 (2016).</li><li>Woodruff, K., Maerkl, S. 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J., 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=Achieving+accurate+compound+concentration+in+cell-based+screening:+validation+of+acoustic+droplet+ejection+technology.">Achieving accurate compound concentration in cell-based screening: validation of acoustic droplet ejection technology.</a> Journal of Biomolecular Screening. 14 (5), 452-459 (2009).</li><li>Sackmann, E. K., 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=Technologies+That+Enable+Accurate+and+Precise+Nano-+to+Milliliter-Scale+Liquid+Dispensing+of+Aqueous+Reagents+Using+Acoustic+Droplet+Ejection.">Technologies That Enable Accurate and Precise Nano- to Milliliter-Scale Liquid Dispensing of Aqueous Reagents Using Acoustic Droplet Ejection.</a> Journal of Laboratory Automation. 21 (1), 166-177 (2016).</li><li>Day, R. N., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluorescent+protein+palette:+tools+for+cellular+imaging.">The fluorescent protein palette: tools for cellular imaging.</a> Chemical Society Reviews. 38 (10), 2887-2921 (2009).</li><li>Zielinski, D., Gordon, A., Zaks, B. L., Erlich, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPipet:+sample+handling+using+a+tablet.">iPipet: sample handling using a tablet.</a> Nature Methods. 11 (8), 784-785 (2014).</li><li>Brunner, 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=Cell+cycle+dependence+of+gene+transfer+by+lipoplex,+polyplex+and+recombinant+adenovirus.">Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus.</a> Gene Therapy. 7 (5), 401-407 (2000).</li><li>Nii, T., 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=Single-Cell-State+Culture+of+Human+Pluripotent+Stem+Cells+Increases+Transfection+Efficiency.">Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency.</a> BioResearch Open Access. 5 (1), 127-136 (2016).</li><li>Noonan, D. J., Henry, K., Twaroski, M. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+dry+DNA+storage+and+shipment+at+room+temperature.">Protocols for dry DNA storage and shipment at room temperature.</a> Molecular Ecology Resources. 13 (5), 890-898 (2013).</li><li>Lesnick, J., Lejeune-Dodge, A., Ruppert, N., Jarman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> High-Precision Cell Dispensing with the Labcyte Echo&#174; Liquid Handler. , (2017).</li><li>Yang, 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=A+public+genome-scale+lentiviral+expression+library+of+human+ORFs.">A public genome-scale lentiviral expression library of human ORFs.</a> Nature Methods. 8 (8), 659-661 (2011).</li><li>Peng, J., Zhou, Y., Zhu, S., Wei, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screens+in+mammalian+cells+using+the+CRISPR-Cas9+system.">High-throughput screens in mammalian cells using the CRISPR-Cas9 system.</a> The FEBS journal. 282 (11), 2089-2096 (2015).</li></ol>

The establishment and optimization of an accurate high-throughput transfection method for a given cell line require scientists to follow some key parameters described in this section. We strongly encourage starting with the recommended values throughout the protocol as these settings optimized for HeLa cells also proved to be efficient for HEK cells. However, as the best parameters may depend on the cell lines and transfection reagents, optimal conditions can be defined by&#160;varying the cell number, diluent volume, to...

The method presented here describes in detail how to perform DNA plasmid multiplexing and transfection in mammalian cells at high throughput using an acoustic-based liquid nanodispenser in a 384-well plate, even for nonspecialists in the field. This recently published method1 allows performing as much as 384 independent plasmid DNA multiplexing and transfection conditions in one experiment, in less than 1 h. Single or cotransfection experiments were successful, reaching a near 100% cotransfection within the transfected cells population. This protocol makes transfection easier because most of the tedious, time-consuming, and error-prone steps are now software driven (see Figure 1 for a general overview). Further efforts have been made to develop dedicated tools to enhance the ease of use while avoiding human errors during the overall process and to promote successful transfection even for nonspecialists in the field. The described protocol includes a &#34;user-friendly&#34; macro spreadsheet that we developed in order to manage 384 independent transfection conditions with multiplexing possibilities of up to four plasmids in each well. The macro automatically generates templates of the source plate(s) to load the expected DNA plasmid volume from starting stock solutions and the files required to drive the nanodispenser software upon the experimental design that has been inputted. As the manual dispensing of DNA in a 384-well source plate is tedious and error-prone, we also developed a dedicated tablet-based application to guide the user while dispensing DNA solution according to the template.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59570/59570fig1v3.jpg" /> 
Figure 1: Experimental workflow. Schematic representation of the optimal automated high-throughput reverse transfection protocol (from experimental design to custom biological assay). Manual steps are indicated by the hand symbol and the approximate time for each step is written in a red box. <a href="https://www.jove.com/files/ftp_upload/59570/59570fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Many cell-based experiments start with plasmid DNA transfection, and even if many dedicated reagents have been and are still being developed to enhance transfection efficiency and/or ease the procedure, much remains to be done2,3,4. DNA plasmid cell transfection involves several steps to reach high efficiency, such as an initial complex uptake, endosomal escape, and cytoplasmic transport to the nucleus5,6. In addition to calcium precipitation or physical techniques such as electroporation or microinjection using dedicated devices7, modern chemical methods have focused on enhancing DNA cell delivery while lowering the cell cytoxicity8,9. The use of lipids or cationic polymers forming liposome-like complexes and, more recently, nonliposomal polymeric chemistry systems has made transfection easier and more efficient10. Despite these developments, cell transfection still requires specific skills to be accurately performed as most of these physical or chemical transfection protocols require scientists to manually prepare each DNA transfection reaction condition, thus impairing the throughput. To circumvent this problem, reverse transfection protocols have been developed using chemical transfection reagents11,12,13, enabling the user to test or combine several plasmids in a faster way. In these protocols, nucleic acid complexes with transfection reagents are formed before seeding the cells on the complexes. However, these reverse protocols are still limited by the manual handling of DNA solutions and by the combination of each of the independent conditions. Although it is feasible to perform them in a 96-well plate format, the DNA preparation and dispenses will be tedious, and there likely will be mistakes. When different amounts of several DNA plasmids are required and multiplexed with each other, cell transfection becomes even harder to achieve and more time-consuming, and human errors become quite inevitable. Scaling up to the 384-well plate format in a reverse transfection approach, in spite of few multiplexed DNA transfection conditions, becomes an impossible challenge due to the following reasons. i) The DNA amounts, transfection reagent, or reaction mixture volumes to manage are lower than 1 &#181;L for each well. ii) The multiplexing of plasmids for 384 independent conditions becomes extremely complicated. The delivery in each of the 384 wells is also iii) highly time-consuming and iv) error-prone. Indeed, dispensing the right solution in the expected wells is hard to manage because the low volumes already dispensed do not allow visual monitoring between the empty and already filled wells. v) Finally, there is a high risk of drying the mixture by evaporation before the cells are added due to the time needed to perform the necessary dispensing steps. In summary, the limiting factor to set up high-throughput DNA plasmid transfection assays appears to be the miniaturization of the assay, which implies low-volume multiplexing and managing that cannot be handled manually anymore but are also hardly achievable in a reliable way by classical peristatic liquid handlers.
As a proof of difficulty to automatize such assays and gain high-throughput, only a few attempts to automate transfection have been published so far: a 96-well plate format using a commercial liquid handling device and calcium phosphate precipitation14 and, more recently, a lipoplex reagent, and a microfluidic chip enabling 280 independent transfections15 but requiring specialized skills in this field. Another method, acoustophoresis, allowing liquid levitation and leading to fluid manipulation and mixing, was used to perform DNA transfection in 24- to 96-well plate formats16. Although feasible, this approach suffers from an extremely low throughput as the mixing of cells with DNA transfection mixture requires a 60 s incubation for every single point before seeding. This implies a duration of at least 96 min for a complete 96-well plate. Furthermore, this protocol is far away from being amenable to the overall biologists&#39; audience as this work was done with an in-house designed and manufactured device which is currently not available on the market. On the contrary, in the last few years, an easy to use software-driven acoustic-based dispensing technology has emerged with nanovolume dispenser devices. Using focused acoustic energy, these devices allow the tightly controlled ejection of small liquid volumes from 2.5 nL to 500 nL from a source plate to a destination one17. This technology, called acoustic droplet ejection (ADE), has numerous advantages: it is fully automated, contactless, tipless, accurate, precise, and highly reproducible, and it has a high throughput18. First devoted to delivering dimethyl sulfoxide (DMSO) solutions, settings have been enhanced to dispense aqueous buffers19. Acoustic nanodispensers, then, seem suitable for reverse cell transfection protocols and could circumvent most of the above-mentioned manual limitations. As no plasmid transfection attempts were previously described using this technology, we recently evaluated the suitability of an acoustic-based dispensing system to perform reverse cell transfection.
Taking advantage of the nanodispenser throughput and ease of use, we optimized a reverse transfection protocol for HeLa cells by cross-testing several parameters that can influence DNA transfection on a 384-well, single plate, namely, the total DNA amount and source DNA starting concentration, diluent volume, transfection reagent, and number of spread cells. The developed protocol circumvents the above-described manual limitations of cell transfection and presents several advantages over other automated transfection attempts. First, it is miniaturized, thus allowing for cost-effective transfection reagent by saving DNA plasmid preparations and transfection reagent. Secondly, it is much more high-throughput and reproducible than the manual protocol (even for beginners), as transfection of an entire 384-well plate can be achieved in less than 1 h. Finally, it is software-driven, allowing the control of the dispensed DNA amount and the multiplexing of several plasmids. Indeed, thanks to the nanodispenser software (Table of Materials), the user can elaborate a study plan to control the volumes to be dispensed from a defined source well plate to a destination one.
The protocol presented here is mainly intended for those who have access to a nanodispenser and would like to set up transfection experiments at high throughput, but also for those who want to rapidly optimize their transfection parameters for a given cell type by applying this protocol to cross-test several parameters at high throughput. Indeed, we have shown that optimized parameters identified with this nanoscale protocol can be transposed to larger-scale and manual transfection experiments. Finally, as the transfection reagent used in the present protocol allows DNA or siRNA transfection according to the manufacturer, the protocol is also of interest to those aiming at performing array approaches for gene overexpression or knockdown. The destination plates prefilled with DNA can be conserved up to 7 days before use in a transfection assay without loss of efficacy, which is another advantage of the following protocol for this kind of application.

<table border="0" cellpadding="0" cellspacing="0" style="width:1226px;" width="1224">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">384LDV Microplate</td>
			<td style="width:363px;">Labcyte</td>
			<td style="width:202px;">LP-0200</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">384-well Microplate &#956;Clear Black</td>
			<td style="width:363px;">Greiner</td>
			<td style="width:202px;">781906</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">Ampicilin</td>
			<td style="width:363px;">Sigma</td>
			<td style="width:202px;">A9393-5G</td>
			<td style="width:291px;">Selection antibiotic for bacteria transformed with ampicilin expressing vector</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Android Tablet</td>
			<td style="width:363px;">Samsung</td>
			<td style="width:202px;">Galaxy Note 8</td>
			<td style="width:291px;">used to guide the user while the source plate manual dispense</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Aniospray Surf 29</td>
			<td style="width:363px;">Anios</td>
			<td style="width:202px;">2421073</td>
			<td style="width:291px;">disinfectant to clean the MicroFlo head</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Columbus software</td>
			<td style="width:363px;">Perkin Elmer</td>
			<td style="width:202px;"></td>
			<td style="width:291px;">image analysis software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Dulbecco&#39;s Modified Eagle Medium (DMEM), high glucose, GlutaMAX Supplement, pyruvate</td>
			<td style="width:363px;">Thermo Fisher Scientific</td>
			<td style="width:202px;">10566032</td>
			<td style="width:291px;">cell culture medium</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:371px;">Echo Cherry Pick 1.5.3 software</td>
			<td style="width:363px;">Labcyte</td>
			<td style="width:202px;"></td>
			<td style="width:291px;">Software enabling ADE-based dispenses by the Echo550 device from a *.csv file; nanodispenser software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Echo550</td>
			<td style="width:363px;">Labcyte</td>
			<td style="width:202px;"></td>
			<td style="width:291px;">ADE-based dispenser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Fetal Bovine Serum</td>
			<td style="width:363px;">Thermo Fisher Scientific</td>
			<td style="width:202px;">16000044</td>
			<td style="width:291px;">to add in cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:363px;">Sigma-Aldrich</td>
			<td style="width:202px;">HT501128-4L</td>
			<td style="width:291px;">to fix cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">HeLa cells</td>
			<td style="width:363px;">ATCC</td>
			<td style="width:202px;">HeLa (ATCC&#174; CCL-2&#8482;)</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Hoechst 33342, Trihydrochloride, Trihydrate</td>
			<td style="width:363px;">Thermo Fisher Scientific</td>
			<td style="width:202px;">H3570</td>
			<td style="width:291px;">10 mg/mL Solution in Water</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">INCell Analyzer 6000</td>
			<td style="width:363px;">GE Healthcare</td>
			<td style="width:202px;">29043323</td>
			<td style="width:291px;">automated laser-based confocal imaging platform</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:371px;">LB medium</td>
			<td style="width:363px;">Thermoischer Scientific 
			LB Broth Base (Lennox L Broth Base)&#174;, powder</td>
			<td style="width:202px;">12780052</td>
			<td style="width:291px;">culture medium for bacteria growth</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">Lysis Buffer (A2)&#160;</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740912.1</td>
			<td style="width:291px;">Buffer from the NucleoSpin Plasmid kit used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">MicroFlo 10&#181;L cassette</td>
			<td style="width:363px;">Biotek Instruments Inc</td>
			<td style="width:202px;">7170013</td>
			<td style="width:291px;">to use with the Microflo Dispenser</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">MicroFlo 1&#956;L cassette</td>
			<td style="width:363px;">Biotek Instruments Inc</td>
			<td style="width:202px;">7170012</td>
			<td style="width:291px;">to use with the Microflo Dispenser</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">MicroFlo Dispenser</td>
			<td style="width:363px;">Biotek Instruments Inc</td>
			<td style="width:202px;">7171000</td>
			<td style="width:291px;">peristaltic pump-based liquid handler device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Microvolume spectrophotometer</td>
			<td style="width:363px;">Denovix</td>
			<td style="width:202px;">DS-11 Spectrophotometer</td>
			<td style="width:291px;">Measure the DNA concentration of samples</td>
		</tr>
		<tr height="86">
			<td height="86" style="height:86px;width:371px;">mVenus plasmid</td>
			<td style="width:363px;">mVenus cDNA was cloned by enzymatic restriction digestion and ligation in Age1/BsrG1 sites of the tdTomato-N1 plasmid</td>
			<td style="width:202px;"></td>
			<td style="width:291px;">Vector type: Mammalian Expression, Fusion Protein: mVenus</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">Neutralization Buffer (A3)&#160;</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740913.1</td>
			<td style="width:291px;">Buffer from the NucleoSpin Plasmid kit used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">NucleoSpin Plasmid kit</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740588.50</td>
			<td style="width:291px;">used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Optimal-Modified Eagle Medium (Opti-MEM) Medium</td>
			<td style="width:363px;">Thermo Fisher Scientific</td>
			<td style="width:202px;">31985070</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">optional Wash bufferWash Buffer (A4)</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740914.1</td>
			<td style="width:291px;">Buffer from the NucleoSpin Plasmid kit used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:50px;">orbital shaker&#160;</td>
			<td style="width:363px;">incubated large capacity shaker</td>
			<td style="width:202px;">444-7084</td>
			<td style="width:291px;">Used to grow bacteria under gentle agitation and 37&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Penicillin-Streptomycin</td>
			<td style="width:363px;">Thermo Fisher Scientific</td>
			<td style="width:202px;">15140122</td>
			<td style="width:291px;">10,000 U/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Phosphate-Buffered Saline</td>
			<td style="width:363px;">Thermo Fisher Scientific</td>
			<td style="width:202px;">10010001</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">Plasmid mini-columns</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740499.250</td>
			<td style="width:291px;">Silica membrane mini-column to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">Resuspension Buffer (A1)&#160;</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740911.1</td>
			<td style="width:291px;">Buffer from the NucleoSpin Plasmid kit used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">RNAse A</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740505</td>
			<td style="width:291px;">Enzyme from the NucleoSpin Plasmid kit used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">tdTomato-N1 plasmid</td>
			<td style="width:363px;">Addgene</td>
			<td style="width:202px;">Plasmid #54642</td>
			<td style="width:291px;">Vector type: Mammalian Expression, Fusion Protein: tdTomato&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">TransIT-X2 Dynamic Delivery System</td>
			<td style="width:363px;">Mirus Bio</td>
			<td style="width:202px;">MIR 6000</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">Wash Buffer (AW)</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740916.1</td>
			<td style="width:291px;">Buffer from the NucleoSpin Plasmid kit used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">3D printer</td>
			<td style="width:363px;">Creality</td>
			<td style="width:202px;">CR10S</td>
			<td style="width:291px;">used to print the plate adapter</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:371px;">Blender Software&#160;</td>
			<td style="width:363px;">&#160;https://www.blender.org/ 
			Free software under GNU General Public License (GPL).</td>
			<td style="width:202px;">version 2.79b</td>
			<td style="width:291px;">used to design the plate adapter</td>
		</tr>
	</tbody>
</table>

high-throughput dna plasmid multiplexing and transfection using acoustic nanodispensing technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

fIn order to determine if the ADE technology could be used for an automated reverse transfection protocol, we monitored cell transfection efficiency by fluorescence microscopy, using a red fluorescent tdTomato expressing plasmid. First aiming at determining the best transfection parameters, different diluent volumes and total amounts of DNA were cross-tested. Diluent volume was used to allow the DNA droplets, once dispensed, to spread all over the wells to circumvent inhomogeneous transfe...

Watch this Scientific Journal Video about High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology at JoVE.com

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

The NanoDispenser based transfection protocol on developed to describe in this video represents the first user-friendly, high-throughput transfection method allowing even beginners in the field to be successful. This is easy on high-throughput technique allows transfection of cell with 384 independent condition. As of now, the method is low cost due to the nano volume of reagent needed.To define the best transfection parameters for all the cell type, we suggest keeping the source DNA concentration constant, and using varying amounts of transfected DNA, transfection reagent, and number of cells. For dispensing the 40 microliter cell suspension, mount a 10 microliter cassette on the peristaltic liquid handler device, and prepare an appropriate program. First, adjust the flow rate parameter to low to dispense cells with a low speed to avoid promoting potential damage to the cells by sheer stress and high impact on the bottom of the wells.Then, adjust the dispensation height to 11.43 millimeters. The height must be high enough to lower the cell impact on the bottom of the wells during the dispensing process, but low enough to avoid retention of the droplets on the dispensing head. Next, adjust the plate clear height to 16 millimeters to allow free displacement of the dispensing head over the plate after dispensing each row.Visually control the proper settings of the peristaltic liquid handler head height. Make sure no drops are retained on the dispensing tips while dispensing. Also verify that the head is high enough to allow displacement of the head after dispensing each row.To generate picklists to drive the ADE-dispensation, a user-friendly spreadsheet macro was developed to manage DNA amounts and mix up to four plasmids in a 384 well plate format. Optimal volumes are pre-filled in some cells of the spreadsheet, but can be changed. In the pink fields, the transfection reagent, or TR mixture value, is set to 500 nanoliters.The minimal volume value in the source plate wells is set to four microliters, and the maximal volume in the source plate wells to 11.25 microliters. Enter 100 nanograms per microliter DNA starting concentrations in the blue fields corresponding to the underlying DNA. Then, enter the desired DNA amount in the gray and green fields corresponding to the 384 wells of the plate.Enter the amounts and plasmid names and ensure the same spelling is used if the same plasmid has to be transfected in several wells. Click on generate picklists to verify the entered values, and if requested, correct the orange-filled cells indicating errors or volumes that cannot be handled by the NanoDispenser. If none are detected, the macro will generate the 384 well dispensing guide, the DNA picklist file, and the TR picklist file from data collected on the corresponding sheets.Print the template from the source plate sheet to visualize the wells to be filled before dispensing. Plasmid names and minimal fill volumes are indicated. Transfection reagent mixture volumes that will be filled in the following wells, are indicated as TR and highlighted in green.To prepare the DNA source plate, dilute the stored DNA plasmid to 100 nanograms per microliter using distilled water. Now, calibrate the 384 well grid to the plate dimensions. Open the 384 Well Pipetting Guide Application on a tablet.Place the source place on the grid on the lower screen. In the upper left calibration menu, click plus or minus to enhance or reduce the size of the grid and wells in order to adjust the green wells to the four corner wells of the plate. Using double sided tape, mount the 3D printed plate adapter on the screen to avoid source plate movements while dispensing.If needed, move the calibrated grid using the rotation arrows and up, down, right, left buttons to adjust the grid to the plate position. Once the grid and well sizes are properly calibrated and located, tick the lock calibration box. Click on file and open the 384-Wells-Pipetting-Guide.csv file. Follow the screen instructions to manually dispense the indicated volume of plasmid at the specified concentration into the white highlighted well that corresponds to the proper target destination of the expected plate. Use minus or plus arrows to go back or further in the DNA dispensing process.Stop dispensing upon reaching the first transfection reagent solution to load. Once the DNA dispensations are finished, remove the source plate from the adapter. If several source plates are to be filled, place a new source plate on the adapter and follow the dispensing instructions.Centrifuge the DNA filled source plates to ensure proper liquid leveling and to remove bubbles leading to inaccuracy in the ADE base transfers. Now, run a survey to control the manually dispensed volumes. On the NanoDispenser PC, run the NanoDispenser program.Go to the diagnostic tab, tick the source plate outbox, load the source plate on the plate holder and tick in to enter the plate. When prompted, select 384LDV_AQ_B2 to set the NanoDispenser to the aqueous buffer dispensing mode and press OK.Select survey in the miscellaneous menu. And click on launch.Select the prefilled wells to analyze and click on the go button. Verify that the measured volumes match the expected ones and ensure no wells have been loaded with volumes of more than 12 microliters. Prime the tubing with serum free medium by filling a new sterile vessel with 10 milliliters of pre-warmed serum free medium and submerging the tube organizer in it.Press the prime button of the peristaltic liquid handler for about 10 seconds. Make sure the tip is not clogged by visually inspecting the flow from the dispensing head. Place a sterile 384 well culture plate on the peristaltic liquid handler plate carrier, and remove its lid.Run the pre-calibrated program to dispense one microliter in each well of the 384 well plate. The dispensing time is approximately eight seconds. Then replace the lid of the 384 well plate.To set up ADE driven DNA dispensing, run the picklist software. Set the 384 wells sourced to 384LDV. Set the device to aqueous buffer dispensing mode by selecting 384LDV_AQ_B2.And the destination plate types to Greiner_384PS_781096. Un-tick optimized transfer throughput. Select pick list tab.Click on import. And select the DNA_Picklist_CSV file. Then, click on play and save the protocol.Click on simulate to perform a simulation of the program dispensations to make sure that the picklist matches the expected experimental design. Click on the run button and start the dispensing program. When asked, insert the requested source plate.And then the destination plate in the NanoDispenser. To set up the transfection reagent dispensation, work in a biosafety cabinet to dilute lipopolyplex transfection reagent in serum free medium to a one x final concentration and vortex the tube. Immediately dispense the TR mix according to the predefined source plate deigned by the macro and using the pre-calibrated 384 well pipetting guide application.Following TR dispensing, perform a survey to control the TR loaded volumes as done before for the loaded DNA. Select the transfection reaction mix prefilled wells to analyze. And click on the go button.Verify that the measured volumes match the expected ones. And ensure no wells have been loaded with volumes of more than 12 microliters. Now perform a reset of the DNA picklist in the picklist software.Verify that the device parameters are still set to aqueous buffers. Enter the correct source and destination plate types. On the pick list tab, click on reset to clear the sample list.Then click on import and choose the TR_Picklist. CSV file. Click on play.Save the protocol if prompted and perform a simulation of the program transfection reagent mixture dispensations to ensure proper design by clicking on the simulate button. As done previously, after clicking on the run button and starting the dispensing program place the source plate and then the destination plate in the NanoDipsenser as requested. To dispense the cells, fill a new sterile vessel with the prepared cell suspension and stir it to avoid sedimentation leading to inaccuracy and cell density.Insert the tube organizer in the solution. Then, press the prime button until the cell suspension starts to flush from the dispensing head. Make sure the tip is not clogged by visually inspecting the flow from the dispensing head while flushing and ensure each tube is loaded with cell suspension.Now, load the DNA and TR filled 384 well destination plate on the peristaltic liquid handler plate carrier and remove its lid. Run the pre-calibrated program to dispense 40 microliters of the cell suspension on the complete 384 well plate. The dispensing time is about 45 seconds.Finally, replace the lid of the 384 well plate. The transfection of HeLa cells using lipopolyplex reagent was successful. DNA amounts ranging from five to 30 nanograms showed the same efficiency and up to 90%cell transfection at one microliter diluent volume.In contrast, higher amounts resulted in an abrupt decrease in percentage of transfected cells. Various diluent volumes ranging from 15 nanoliters to four microliters were tested and one microliter was identified as the best condition. To further enhance the throughput of this protocol two efficient DNA storage methods were tested namely dry storing the plate or frozen storage.Both storage methods did not lead to significantly different results than freshly dispensed DNA solution stored for up to seven days. As plasma transfection usually employs at least two different plasmids, the DNA multiplexing ability of this protocol was examined using the best identified conditions. The previously used tdTomato red fluorescent protein expressing plasmid was modified to express mVenus a bright yellow fluorescent protein and both were then used in cotransfection attempts.Red or green fluorescent positive cell analysis showed the transfection efficiency to be about 80%However, nearly 100%of the red cells were also cotransfected with the mVenus expressing plasmid as can be seen in the representative software based image analysis. Once DNAs have been dispensed in the source plate. Perform a survey to ensure that the expected volumes have been loaded and do not exceed 12 microliters.While dispensing the transfection reagent in the source plate, try to avoid bubbles as centrifuging sub plates will result in a complete loss of transfection efficacy. This method can be applied to biological experiment, crimes, and transfection and is suitable for mostly that can be performed in a 384 well plate format. A fullproof transfection paves the way for new applications such as plasmid based known for crisper cast non-screening approaches enabling the monitoring of intermittent affect currently unable to be sorted in poor strategies.

high-throughput-dna-plasmid-multiplexing-transfection-using-acoustic

Research

JoVE Journal

Genetics

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 ...

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 ...

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 ...

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring fitness phenotypes and improving our understanding of how genotypes are shaped by selection. Extensive recent efforts to develop and apply niche continuous culture devices have revealed the benefits of conducting new forms of cell culture control. This includes defining custom selection pressures and increasing throughput for studies ranging from long-term experimental evolution to genome-wide library selections and synthetic gene circuit characterization. The eVOLVER platform was recently developed to meet this growing demand: a continuous culture platform with a high degree of scalability, flexibility, and automation. eVOLVER provides a single standardizing platform that can be (re)-configured and scaled with minimal effort to perform many different types of high-throughput or multi-dimensional growth selection experiments. Here, a protocol is presented to provide users of the eVOLVER framework a description for configuring the system to conduct a custom, large-scale continuous growth experiment. Specifically, the protocol guides users on how to program the system to multiplex two selection pressures &#8212; temperature and osmolarity &#8212; across many eVOLVER vials in order to quantify fitness landscapes of Saccharomyces cerevisiae mutants at fine resolution. We show how the device can be configured both programmatically, through its open-source web-based software, and physically, by arranging fluidic and hardware layouts. The process of physically setting up the device, programming the culture routine, monitoring and interacting with the experiment in real-time over the internet, sampling vials for subsequent offline analysis, and post experiment data analysis are detailed. This should serve as a starting point for researchers across diverse disciplines to apply eVOLVER in the design of their own complex and high-throughput cell growth experiments to study and manipulate biological systems.

The eVOLVER framework enables high-throughput continuous microbial culture with high resolution and dynamic control over experimental parameters. This protocol demonstrates how to apply the system to conduct a complex fitness experiment, guiding users on programming automated control over many individual cultures, measuring, collecting, and interacting with experimental data in real-time.

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring ...

Single-Molecule F&ouml;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule F&#246;rster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.

Single-Molecule F&amp;#246;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Presented here is a protocol for performing single-molecule F&#246;rster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.