Name: purification of low-abundant cells in the drosophila visual system
Uploaded: 2018-09-26T15:00:00
Description: Watch this Scientific Journal Video about Purification of Low-abundant Cells in the Drosophila Visual System at JoVE.com

This research was funded by the NINDS of the National Institutes of Health under award number K01NS094545, andgrants from the Lefler Center for the Study of Neurodegenerative Disorders. We acknowledge Liming Tan and Jason McEwan for valuable conversations.

1. Planning Before the Experiment
<ol>
	<li>Before starting the experiment, perform a small-scale pilot experiment to confirm if the genetics work as expected to specifically label the desired cells.
	<ol>
		<li>As a first pass, use immunohistochemistry and light microscopy to determine if unwanted cells are labeled. Ideally, genetic markers will be specific within the retina and optic lobe (Figure 3). Only optic lobes are used for cell dissociation and so labeling in the ...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+analysis+of+pure+cell+populations+from+Drosophila.">Preparation and analysis of pure cell populations from Drosophila.</a> Methods in Cell Biology. 44, 143-159 (1994).</li><li>Bryant, 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=Characterization+of+differentially+expressed+genes+in+purified+Drosophila+follicle+cells:+toward+a+general+strategy+for+cell+type-specific+developmental+analysis.">Characterization of differentially expressed genes in purified Drosophila follicle cells: toward a general strategy for cell type-specific developmental analysis.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (10), 5559-5564 (1999).</li><li>Neufeld, T. P., de la Cruz, A. F., Johnston, L. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-activated+cell+sorting+(FACS)+of+Drosophila+hemocytes+reveals+important+functional+similarities+to+mammalian+leukocytes.">Fluorescence-activated cell sorting (FACS) of Drosophila hemocytes reveals important functional similarities to mammalian leukocytes.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (9), 2912-2917 (2004).</li><li>Bryantsev, A. L., Cripps, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+cardiac+cells+from+Drosophila+embryos.">Purification of cardiac cells from Drosophila embryos.</a> Methods. 56 (1), 44-49 (2012).</li><li>Harzer, H., Berger, C., Conder, R., Schmauss, G., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FACS+purification+of+Drosophila+larval+neuroblasts+for+next-generation+sequencing.">FACS purification of Drosophila larval neuroblasts for next-generation sequencing.</a> Nature Protocols. 8 (6), 1088-1099 (2013).</li><li>Khan, S. J., Abidi, S. N., Tian, Y., Skinner, A., Smith-Bolton, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+gentle+and+scalable+method+for+dissociation+and+fluorescent+sorting+of+imaginal+disc+cells+for+mRNA+sequencing.">A rapid, gentle and scalable method for dissociation and fluorescent sorting of imaginal disc cells for mRNA sequencing.</a> Fly (Austin). 10 (2), 73-80 (2016).</li><li>Picelli, 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=Full-length+RNA-seq+from+single+cells+using+Smart-seq2.">Full-length RNA-seq from single cells using Smart-seq2.</a> Nature Protocols. 9 (1), 171-181 (2014).</li><li>Nern, A., Zhu, Y., Zipursky, S. 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This protocol is simple and not technically difficult to execute, but there are several key steps that if overlooked will cause a considerable reduction in cellular yield. (Step 2.3.2.) It is crucial that crosses are healthy, and that the food does not dry out. Regular watering of crosses is essential to maximize the number of flies available for dissection that are of the right genotype and at the correct stage of development. How often crosses need to be watered will vary depending on the food used and the housing cond...

The Drosophila visual system is an outstanding model for studying the genetic basis of development and behavior. It comprises a stereotyped cellular architecture1 and an advanced genetic toolkit for manipulating specific cell types2,3. A major strength of this system is the ability to autonomously interrogate gene function in cell types of interest with single cell resolution, using genetic mosaic methods4,5. We sought to combine these genetic tools with recent advances in next generation sequencing to perform cell type-specific transcriptomic and genomic analyses in single cell clones in genetic mosaic experiments.
To accomplish this, it is essential to develop a robust and efficient method to selectively isolate low abundant cell populations in the brain. Previously, we developed a protocol for isolating specific cell types in the visual system during pupal development through FACS, and determining their transcriptomes using RNA-seq6. In these experiments, the vast majority of cells of a particular type (e.g. R7 photoreceptors) were fluorescently labeled. Using this method, in genetic mosaic experiments, wherein only a subset of cells of the same type are labeled, we failed to isolate enough cells to obtain quality sequencing data. To address this, we sought to increase the cellular yield by improving the cell dissociation protocol.
Our approach was to decrease the total length of the protocol to maximize the health of dissociated cells, improve cellular health by altering dissection and dissociation buffers, and reduce the amount of mechanical disruption to the dissected tissue. We tested the improved protocol7 using mosaic analysis with a repressible cell marker5 (MARCM), which allows generation of single fluorescently labeled clones of particular cell types, that are wild type or mutant for a gene of interest in an otherwise heterozygous fly. Where, under identical conditions, our earlier protocol failed to generate enough material for RNA-seq, the improved protocol was successful. We reproducibly achieve a high cellular yield (~25% of labeled cells) and obtain high quality RNA-seq data from as little as 1,000 cells7.
A number of protocols have been previously described to isolate particular cell types in Drosophila6,8,9,10,11,12,13,14,15,16. These protocols are mostly intended for isolating cells that are abundant within the brain. Our protocol is optimized for isolating low abundant cell populations (fewer than 100 cells per brain) in the visual system using FACS for subsequent transcriptomic and genomic analyses. With this protocol, we aim to provide a way to reproducibly isolate low abundant cells from the fly visual system by FACS and obtaining high quality transcriptome data by RNA-seq.

<table>
	<tbody>
		<tr>
			<td>Liberase TM</td>
			<td>Roche</td>
			<td>5401127001</td>
			<td>Proteolytic enzyme blend</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G0350500</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutathione</td>
			<td>Sigma-Aldrich</td>
			<td>G6013</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Inactivated Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Solution</td>
			<td>Sigma-Aldrich</td>
			<td>I0516</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4458</td>
			<td></td>
		</tr>
		<tr>
			<td>Schneider&#39;s Culture Medium</td>
			<td>Gibco</td>
			<td>21720024</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>LK003178</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M6250-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Micro Kit</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td>RNA purification kit</td>
		</tr>
		<tr>
			<td>RNase-free DNase</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase</td>
			<td>Life Technologies</td>
			<td>18064-014</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP Mix</td>
			<td>Life Technologies</td>
			<td>R0191</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 Solution</td>
			<td>Sigma-Aldrich</td>
			<td>M1028-10X1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Betaine Solution</td>
			<td>Sigma-Aldrich</td>
			<td>B0300-1VL</td>
			<td></td>
		</tr>
		<tr>
			<td>RNaseOUT</td>
			<td>Life Technologies</td>
			<td>10777-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Q5 High-Fidelity 2x Master Mix</td>
			<td>New England Biolabs</td>
			<td>M0492S</td>
			<td></td>
		</tr>
		<tr>
			<td>MinElute PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28004</td>
			<td></td>
		</tr>
		<tr>
			<td>Nextera XT DNA Library Prepration Kit</td>
			<td>Illumina</td>
			<td>FC-131-1024</td>
			<td></td>
		</tr>
		<tr>
			<td>Nextera XT Index Kit</td>
			<td>Illumina</td>
			<td>FC-131-1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Test Tube with Cell Strainer Snap Cap</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle-Top Vacuum Filter Systems</td>
			<td>Corning</td>
			<td>CLS431153</td>
			<td></td>
		</tr>
		<tr>
			<td>ThermoMixer F1.5</td>
			<td>Eppendorf</td>
			<td>5384000020</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSAria Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>656700</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq 2500 Sequencing System</td>
			<td>Illumina</td>
			<td>SY&#8211;401&#8211;2501</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification of low-abundant cells in the drosophila visual system

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small numbers of cells. Utilizing this technology to study development in the Drosophila visual system, which boasts a wealth of cell type-specific genetic tools, provides a powerful approach for addressing the molecular basis of development with precise cellular resolution. For such an approach to be feasible, it is crucial to have the capacity to reliably and efficiently purify cells present at low abundance within the brain. Here, we present a method that allows efficient purification of single cell clones in genetic mosaic experiments. With this protocol, we consistently achieve a high cellular yield after purification using fluorescence activated cell sorting (FACS) (~25% of all labeled cells), and successfully performed transcriptomics analyses on single cell clones generated through mosaic analysis with a repressible cell marker (MARCM). This protocol is ideal for applying transcriptomic and genomic analyses to specific cell types in the visual system, across different stages of development and in the context of different genetic manipulations.

General scheme 
A general scheme of the protocol is shown in Figure 1. The protocol is divided into three major parts: fly work, sample preparation, sequencing and data analysis. The &#34;Planning before the experiment&#34; session of the protocol is not included in the general scheme for simplicity.
Timeline 
A calendar of the major parts of the protocol...

Watch this Scientific Journal Video about Purification of Low-abundant Cells in the Drosophila Visual System at JoVE.com

Purification of Low-abundant Cells in the Drosophila Visual System

Here, we present a cell dissociation protocol for efficiently isolating cells present at low abundance within the Drosophila visual system through fluorescence activated cell sorting (FACS).

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small ...

This method can help address key questions in biology, such as how diverse cell types are generated and organized into higher order structures with specific functions. The main advantage of this technique is that it facilitates efficient purification of cells present at a low abundance in the Drosophila visual system for transcriptomic and genomic analysis. On Tuesday of week six of the protocol, 45 minutes before the dissections, retrieve one vial of papain powder from four degrees Celsius and incubate it at room temperature.Next, set aside Complete Schneider's Medium, or CSM, in an Eppendorf tube at room temperature to add to the papain powder later. Five to ten minutes before the dissections, use a syringe to add the room temperature CSM to the vial of papain directly through the cap to a concentration of 100 units per milliliter. To mix, first invert the vial to capture any powder stuck on the inside of the cap and then pipette the contents up and down.After mixing the reagents, bring the water up to 37 degrees Celsius in a beaker in a water bath. Then place the vial in the beaker for 30 minutes to activate the papain and make sure that the vial is not floating. While the papain is incubating, retrieve an aliquot of proteolytic enzyme blend from the freezer and incubate it at room temperature.After 30 minutes of activation, incubate the papain at room temperature. On Tuesday of week six, dissect the staged pupil brains in cold CSM with clean forceps. Dissect as many brains as possible in 10 minutes and pool the brains in a fresh drop of cold CSM.Cut off the optic lobes by pinching at the junction of the optic lobe and the central brain. Next, add the lobes to the bottom of a microtube containing 500 microliters of 1x RS.One microtube for the experimental tissue and one for the negative control tissue. Keep the tubes on ice.Dissect as many lobes as possible in one hour. Pool the dissected optic lobes in a single microtube to eliminate tissue loss during transfer between microtubes. Carefully remove the 1x RS from the microtube with the dissected optic lobes using a P200 pipette, leaving enough solution to cover the lobes.Carefully add 500 microliters of 1x RS to the side of the tube. Let the lobes settle to the bottom of the tube, then pipette up 200 microliters. Expel the mix, observe the lobes moving and allow the lobes to settle again.For the two samples, aliquot 600 microliters of activated, room temperature papain into a microtube. Next, create the dissociation solution. Add 4.2 microliters of room temperature proteolytic enzyme blend into 600 microliters of papain solution to obtain a final concentration of 0.18 WU per milliliter and mix the solution by pipetting it up and down.Then carefully remove the 1x RS from each sample and add 300 microliters of dissociation solution to the lobes. Incubate the samples at 25 degrees Celsius. 1, 000 RPM for 15 minutes in a microtube ThermoMixer.At the 5 and 10 minute time points, stop the ThermoMixer and let the lobes sink to the bottom of the microtube. Pipette up 200 microliters of the solution and mix. After the 15 minute incubation, wait for the lobes to settle to the bottom and carefully remove the dissociation solution from the lobes without disturbing the lobes.Wash the lobes two times with 1x RS.Carefully add 500 microliters of 1x RS without disturbing the lobes. Then carefully pipette up 200 microliters and gently expel the solution. Allow the lobes to sink to the bottom and repeat.Next, wash each sample two times with 500 microliters of CSM. Carefully remove the CSM and add another 200 microliters of CSM to the tube. Using a P200 pipette, disrupt the tissue by pipetting up and down without foaming until the solution is homogenous.Using a P200 pipette, filter the cell suspension through a 30 micrometer mesh cap into a 5 milliliter fax tube on ice. And make sure the entire suspension goes through. Add 500 microliters of CSM to rinse the dissociation microtube.And filter the solution into the fax tube. Finally, carefully take off the cap of the fax tube. Collect the solution underneath the mesh filter with a P200 and add it to the rest of the suspension in the fax tube.Confocal microscopy of immuno stained lobes with antibodies specific against DsRed and GFP as well as the 24B10 antibody as a reference for the Lamina and Medulla neuropils confirmed that L3 is the only cell type labeled by both DsRed and GFP in the optic lobe. Facts data from 100, 000 events were recorded, of which 29.9%of all the events are potential singlets. After doublets in cells with different sizes were gated out based on granularity and size, the remaining single cells, L3 neurons, appeared as tight clusters in P1, which was well-separated from the background cells.Following this procedure, other methods like RNA-seq or ATAC-seq can be performed to address biological questions through the analysis of gene expression or chromatin organization, respectively. This technique considerably advances the ability of researchers to explore the genetic mechanisms underlying the development and function of complex tissues using the Drosophila visual system as a model.

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