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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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 ...

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