This work was supported by grants from the U.S. National Institutes of Health (DA017279, AG033954, DA037743), the Michael J. Fox Foundation, and the Caltech Innovation Initiative funding the Millard and Muriel Jacobs Genetics and Genomics Laboratory at the California Institute of Technology. We thank Brian Williams for optimising the RNA-Seq library protocol and for providing assistance. We thank Henry Amrhein for computational training. We thank Barbara J. Wold for the use of her equipment and laboratory space. We thank Igor Antoshechkin for library sequencing, and for facility management.

NOTE: All experiments were conducted in accordance with the guidelines for care and use of animals provided by the National Institutes of Health, and protocols were approved by the Institutional Animal Care and Use Committee at the California Institute of Technology.
1. Derivation of Primary Dopaminergic Cell Cultures from Embryonic Mouse Brain
<ol>
	<li>Solutions and Culture Medium
	<ol>
		<li>Preparation of Ascorbic Acid Stock Solution
		<ol>
			<li>Weigh out 352&#160;mg of ascorbic acid. ...

<ol>
	<li>Henley, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+by+nicotine+in+dopaminergic+neurons.">Transcriptional regulation by nicotine in dopaminergic neurons.</a> Biochem Pharmacol. 86 (8), 1074-1083 (2013).</li><li>Marinov, G. 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=From+single-cell+to+cell-pool+transcriptomes:+stochasticity+in+gene+expression+and+RNA+splicing.">From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing.</a> Genome Res. 24 (3), 496-510 (2014).</li><li>Kim, D. H., 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+transcriptome+analysis+reveals+dynamic+changes+in+lncRNA+expression+during+reprogramming.">Single-cell transcriptome analysis reveals dynamic changes in lncRNA expression during reprogramming.</a> Cell Stem Cell. 16 (1), 88-101 (2015).</li><li>Ramskold, 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=Full-length+mRNA-Seq+from+single-cell+levels+of+RNA+and+individual+circulating+tumor+cells.">Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells.</a> Nat Biotechnol. 30 (8), 777-782 (2012).</li><li>Gertz, 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=Transposase+mediated+construction+of+RNA-seq+libraries.">Transposase mediated construction of RNA-seq libraries.</a> Genome Res. 22 (1), 134-141 (2012).</li><li>Morris, J., Singh, J. M., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+analysis+of+single+cells.">Transcriptome analysis of single cells.</a> J Vis Exp. (50), (2011).</li><li>Trapnell, 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=Differential+gene+and+transcript+expression+analysis+of+RNA-seq+experiments+with+TopHat+and+Cufflinks.">Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.</a> Nat Protoc. , 562-578 (2012).</li><li>Srinivasan, R., 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=Smoking-Relevant+Nicotine+Concentration+Attenuates+the+Unfolded+Protein+Response+in+Dopaminergic+Neurons.">Smoking-Relevant Nicotine Concentration Attenuates the Unfolded Protein Response in Dopaminergic Neurons.</a> The Journal of Neuroscience. 36 (1), 65-79 (2016).</li><li>Henderson, B. 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=Menthol+Alone+Upregulates+Midbrain+nAChRs,+Alters+nAChR+Subtype+Stoichiometry,+Alters+Dopamine+Neuron+Firing+Frequency,+and+Prevents+Nicotine+Reward.">Menthol Alone Upregulates Midbrain nAChRs, Alters nAChR Subtype Stoichiometry, Alters Dopamine Neuron Firing Frequency, and Prevents Nicotine Reward.</a> J Neurosci. 36 (10), 2957-2974 (2016).</li><li>Gong, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+expression+atlas+of+the+central+nervous+system+based+on+bacterial+artificial+chromosomes.">A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.</a> Nature. 425 (6961), 917-925 (2003).</li><li>Kim, J., Henley, B. M., Kim, C. H., Lester, H. A., Yang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incubator+embedded+cell+culture+imaging+system+(EmSight)+based+on+Fourier+ptychographic+microscopy.">Incubator embedded cell culture imaging system (EmSight) based on Fourier ptychographic microscopy.</a> Biomed Opt Express. (8), 3097-3110 (2016).</li><li>Lammel, 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=Diversity+of+transgenic+mouse+models+for+selective+targeting+of+midbrain+dopamine+neurons.">Diversity of transgenic mouse models for selective targeting of midbrain dopamine neurons.</a> Neuron. 85 (2), 429-438 (2015).</li><li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nat Rev Genet. 10 (1), 57-63 (2009).</li><li>Dryanovski, D. I., 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=Calcium+entry+and+alpha-synuclein+inclusions+elevate+dendritic+mitochondrial+oxidant+stress+in+dopaminergic+neurons.">Calcium entry and alpha-synuclein inclusions elevate dendritic mitochondrial oxidant stress in dopaminergic neurons.</a> J Neurosci. 33 (24), 10154-10164 (2013).</li><li>Kimm, T., Khaliq, Z. M., Bean, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Regulation+of+Action+Potential+Shape+and+Burst-Frequency+Firing+by+BK+and+Kv2+Channels+in+Substantia+Nigra+Dopaminergic+Neurons.">Differential Regulation of Action Potential Shape and Burst-Frequency Firing by BK and Kv2 Channels in Substantia Nigra Dopaminergic Neurons.</a> J Neurosci. 35 (50), 16404-16417 (2015).</li></ol>

Here we isolate single cells from a heterogeneous population using a fluorescent tag, then study each cell with single-cell RNA-seq. We report that 100% of the live TH-eGFP cells that we harvested and sequenced were indeed dopaminergic neurons, based on the presence of the following three DA-related gene transcripts, TH, DDC and&#160;slc6a3. All of the TH-eGFP positive cells expressed these three genes as assessed by RNA-Seq. This was concordant with electrophysiological studies that showed that each TH-eGFP cell also di...

Parkinson&#39;s Disease (PD) is an incurable, age-related neurodegenerative disorder. The cause of this relatively common disorder remains poorly understood. There is widespread neuronal loss throughout the brain, with pronounced neuronal degeneration of dopaminergic neurons in the Substantia Nigra (SNc), leading to diagnostic clinical features of bradykinesia, rigidity and tremor.
Primary mixed cultures containing SNc dopaminergic neurons are especially relevant for Parkinson&#39;s disease. Ventral Tegmental Area (VTA) dopaminergic neurons have been implicated in reward and addiction. Ventral Mesencephalic (VM) primary mixed embryonic cultures contain both SNc and VTA dopaminergic (DA) neurons and GABAergic neurons. VM primary cultures can be useful for neuroprotection assays and to elucidate the selective vulnerability of dopaminergic neurons. There is no reliable way to identify dopaminergic cells in culture based on morphology. Here we develop techniques to identify and harvest single dopaminergic neurons, and construct single-cell, high-yield RNA sequencing libraries.
We report representative RNA transcriptome data for single dopaminergic and GABAergic neurons isolated from midbrain cultures. This protocol can be used for neuroprotection, neurodegeneration, and pharmacological assays to study the effects of various treatments on the DA/GABA transcriptome. Because dopaminergic neurons represent a small minority of the neurons expressed in primary VM cultures, the ability to reliably identify these neurons in living cultures will enable an enhanced range of single-cell studies. These novel techniques will facilitate advances in understanding the mechanisms taking place at the cellular level and may have applications elsewhere in the fields of neuroscience and molecular biology.

<table border="0" cellpadding="0" cellspacing="0" width="921">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:395px;">Papain</td>
			<td style="width:241px;">Worthington Biochemical Corporation&#160;</td>
			<td style="width:108px;">LS003126</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Hanks&#8217; Balanced Salt Solution (HBSS), 1X</td>
			<td style="width:241px;">Gibco&#160;</td>
			<td style="width:108px;">14175-095</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Donor Equine Serum</td>
			<td style="width:241px;">Thermo Scientific&#160;</td>
			<td style="width:108px;">SH30074.03</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:241px;">Sigma&#160;</td>
			<td style="width:108px;">A7030-50G</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Medium: Neurobasal medium</td>
			<td style="width:241px;">Gibco&#160;</td>
			<td style="width:108px;">21103-049</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:395px;">Culture media that contains a stabilized form of L-glutamine, L-alanyl-L-glutamine: GlutaMAX</td>
			<td style="width:241px;">Gibco&#160;</td>
			<td style="width:108px;">35050-061</td>
			<td style="width:177px;">0.5 mM final concentration.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">L-Ascorbic acid</td>
			<td style="width:241px;">Sigma&#160;</td>
			<td style="width:108px;">A7506</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:395px;">B27</td>
			<td style="width:241px;">Gibco&#160;</td>
			<td style="width:108px;">17504-044</td>
			<td style="width:177px;">50 X stock solution, 1 X final concentration.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">35 mm glass bottom dishes</td>
			<td style="width:241px;">MatTek&#160;&#160;</td>
			<td style="width:108px;">P35GC-1.5-10-C</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Poly-L-Ornithine</td>
			<td style="width:241px;">Sigma&#160;</td>
			<td style="width:108px;">P4957</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:395px;">Laminin</td>
			<td style="width:241px;">Sigma&#160;</td>
			<td style="width:108px;">L2020</td>
			<td style="width:177px;">Stored at -20&#176;C in 20 &#181;L aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Deoxyribonuclease I (DNase)</td>
			<td style="width:241px;">Sigma&#160;</td>
			<td style="width:108px;">DN25</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Blue pipette tips</td>
			<td style="width:241px;">Sorenson Bioscience,</td>
			<td style="width:108px;">&#160;Inc. 10130</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">P1000</td>
			<td style="width:241px;"></td>
			<td style="width:108px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">10 mm shallow Petri dish</td>
			<td style="width:241px;">VWR&#160;</td>
			<td style="width:108px;">25384-324</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">37&#176;C Water bath</td>
			<td style="width:241px;"></td>
			<td style="width:108px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">37&#176;C, 5% humidity incubator</td>
			<td style="width:241px;"></td>
			<td style="width:108px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">16% paraformaldehyde (PFA)</td>
			<td style="width:241px;">Electron Microscopy Sciences&#160;</td>
			<td style="width:108px;">15710</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Triton X-100</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:108px;">&#160;X100-500ML</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:395px;">Donkey serum</td>
			<td style="width:241px;">Equitech&#160;</td>
			<td style="width:108px;">SD-0100HI</td>
			<td style="width:177px;">100% stock solution, 1% final concentration.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Standard Pattern surgical scissors</td>
			<td style="width:241px;">Fine Science Tools</td>
			<td style="width:108px;">14000-14</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Forceps - 2x3 teeth</td>
			<td style="width:241px;">Fine Science Tools</td>
			<td style="width:108px;">11022-14</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Forceps - Dumont #55</td>
			<td style="width:241px;">Fine Science Tools</td>
			<td style="width:108px;">11295-51</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Forceps - Dumont #5</td>
			<td style="width:241px;">Fine Science Tools</td>
			<td style="width:108px;">11252-23</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">10X PBS, pH 7.4</td>
			<td style="width:241px;">Gibco&#160;</td>
			<td style="width:108px;">70011-044</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Dulbecco&#39;s Phosphate Buffered solution (DPBS) 1X</td>
			<td style="width:241px;">Gibco</td>
			<td style="width:108px;">14190-144</td>
			<td style="width:177px;">500 ml&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">21 guage needle&#160;</td>
			<td style="width:241px;">BD PrecisionGlide Needle</td>
			<td style="width:108px;">305167</td>
			<td style="width:177px;">100 needles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Micropipette puller</td>
			<td style="width:241px;">Sutter Instrument.</td>
			<td style="width:108px;">model P-87</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Micromanipulator&#160;</td>
			<td style="width:241px;">Sutter Instrument</td>
			<td style="width:108px;">&#160;MP-285</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:395px;">Sequencing Kit: SMARTer Ultra Low RNA Kit for Illumina Sequencing</td>
			<td style="width:241px;">Clontech</td>
			<td style="width:108px;">634936</td>
			<td style="width:177px;">100rnxs,incl Advantage2PCR Kit</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:395px;">oligonucleotide (12 &#181;M): SMARTer IIA oligonucleotide (12 &#181;M)</td>
			<td style="width:241px;"></td>
			<td style="width:108px;"></td>
			<td style="width:177px;">From the SMARTer Kit&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Reverse transcriptase (100 units)</td>
			<td style="width:241px;">SMARTcribe reverse transcriptase</td>
			<td style="width:108px;"></td>
			<td style="width:177px;">From the SMARTer Kit&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Primer 1</td>
			<td style="width:241px;">3&#8217; CDSIIa primer&#160;</td>
			<td style="width:108px;"></td>
			<td style="width:177px;">From the SMARTer Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Primer 2</td>
			<td style="width:241px;">IS PCR primer</td>
			<td style="width:108px;"></td>
			<td style="width:177px;">From the SMARTer Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">50X 2 polymerase mix</td>
			<td style="width:241px;">50X Advantage 2 polymerase mix</td>
			<td style="width:108px;"></td>
			<td style="width:177px;">From the SMARTer Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">10X PCR buffer</td>
			<td style="width:241px;">10X Advantage 2 PCR buffer</td>
			<td style="width:108px;"></td>
			<td style="width:177px;">From the SMARTer Kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">RNA Spikes</td>
			<td style="width:241px;">ThermoFisher</td>
			<td style="width:108px;">4456740</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">PCR cooler rack</td>
			<td style="width:241px;">IsoFreeze PCR cooler rack.</td>
			<td style="width:108px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">DNA Sample Preparation Kit&#160;</td>
			<td style="width:241px;">(Illumina/Nextera) Nexter</td>
			<td style="width:108px;">FC-121-1030</td>
			<td style="width:177px;">24 Samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">PCR master mix</td>
			<td style="width:241px;">Nextera PCR master mix (NPM)</td>
			<td style="width:108px;"></td>
			<td style="width:177px;">From the Nextera Kit&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">PCR primer cocktail (PPC)&#160;</td>
			<td style="width:241px;">Nextera PCR primer cocktail (PPC)&#160;</td>
			<td style="width:108px;"></td>
			<td style="width:177px;">From the Nextera Kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Fluorometer</td>
			<td style="width:241px;">The Qubit ThermoFisher</td>
			<td style="width:108px;">Q33216</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">HS DNA BioAnalyzer kit</td>
			<td style="width:241px;">Agilent</td>
			<td style="width:108px;">5067-4626</td>
			<td style="width:177px;">110rxns</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Electrophoresis system&#160;</td>
			<td style="width:241px;">Agilent 2100 Bioanalyzer.</td>
			<td style="width:108px;">G2938C</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Magnetic beads: Agencourt AMPure&#160;</td>
			<td style="width:241px;">Beckman Coulter</td>
			<td style="width:108px;">A63880</td>
			<td style="width:177px;">5ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">RNAse wipe: RNAse Zap wipes</td>
			<td style="width:241px;">Ambion</td>
			<td style="width:108px;">AM9786</td>
			<td style="width:177px;">Size 100 Sheets</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Qubit ds HS Assay Kit</td>
			<td style="width:241px;">Molecular probes&#160;</td>
			<td style="width:108px;">Q32854</td>
			<td style="width:177px;">500 assays</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Gel extraction kit: Qiaquick Gel Extraction Kit</td>
			<td style="width:241px;">Qiagen&#160;</td>
			<td style="width:108px;">28704</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Glass capillary tubing</td>
			<td style="width:241px;">(Kimax-51, 1.5&#8211;1.8 mm o.d.)</td>
			<td style="width:108px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Microforge</td>
			<td style="width:241px;">Narishige (or equivalent)&#160;</td>
			<td style="width:108px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Sequencer</td>
			<td style="width:241px;">Illumina HiSeq instrument</td>
			<td style="width:108px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:395px;">GENSAT tyrosine hydroxylase-eGFP mouse strain&#160;</td>
			<td style="width:241px;"></td>
			<td style="width:108px;">MMRRC stock number: 000292-UNC</td>
			<td style="width:177px;">Stock Tg(Th-EGFP)DJ76Gsat/Mutant Mouse Regional Research Center</td>
		</tr>
	</tbody>
</table>

reliable identification of living dopaminergic neurons in midbrain cultures using rna sequencing and th-promoter-driven egfp expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a tyrosine hydroxylase promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their RNA transcriptome using RNA-seq (Figure 1). The data showed that 100% of the TH-eGFP-positive cells that were harvested and sequenced were dopaminergic neurons, based on the presence of the following three DA-related gene transcripts, TH, dopa decar...

Watch this Scientific Journal Video about Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression at JoVE.com

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

The overall goal of this procedure is to reliably identify living dopaminergic neurons in midbrain cultures using tyrosine hydroxlase driven eGFP expression, single cell harvesting, and RNA sequencing library generation. This method can help answer key questions in the Parkinson's disease and drug addiction fields, because it makes it possible to perform gene expression and electrophysiological assays on identified living dopaminergic neurons in culture. The advantage of this technique is that it provides a means to reliably identify living rather than fixed dopaminergic neurons in primary ventral mesencephalic cultures.Demonstrating the first part of the procedure will be Charlene Kim, a technician from Henry Lester's laboratory. Begin by stabilizing the head of a mouse embryo with forceps firmly placed on the snout, so that the dorsal surface is accessible. Using forceps held in the other hand pinch the layer of skin and the skull just before the ridge of the mesencephalon and peel back the skin and the skull caudelly along the midline.Place the forceps around the ridge with one tip between the cortex and mesoncephalon and the other over the cerebellum. Pinch and remove the entire midbrain. Place the midbrain in a Petri dish with cold HBSS under a dissecting microscope.Under the dissecting microscope, flip the brain segment so that the ventral side is now accessible. There are now four quadrants visible. Remove all of the meninges and vasculature by gently grasping the meninges with forceps and pulling upward away from the brain.Next, place one tong of forceps into the ventricle approximately at the center of the segment. Then to separate the midbrain, make a pinch dorsally, and then pinch medially on each side. Take the lower two quadrants by making laterally cuts on both sides.The ventral tegmental area and the substantia nigra pars compacta are in the ventral inferior section, which appears dense. Place the dissected tissue containing both the VTA and SNC in fresh HBSS in a separate area of the Petri dish. After dissecting all the brain segments, use forceps and a number 11 scalpel to quarter each midbrain section into pieces of approximately equal size.Then, use a wide bore P1000 pipette tip to transfer all of the quartered midbrain segments, to a 15 milliliter conical tube. After allowing the tissue to settle and removing the HBSS add 500 microliters of papain solution. Incubate in a 37 degree Celsius water bath for 15 minutes.Following the incubation, use a wide bore P1000 tip to transfer only the midbrain segments into a one milliliter aliquot of DNase I solution and allow the segments to settle to the bottom of the tube. Then, transfer only the midbrain segments to a 15 milliliter tube containing one milliliter of cold stop solution. Replace the second rinse with one milliliter of fresh stop solution and pipette up and down seven times with a P1000 pipette tip to triturate the cells.Then underlay the cell suspension by slowly pipetting 200 microliters of 4%BSA solution into the bottom of the tube. After centrifugation at 280 x g for six minutes, aspirate the supernatant with a P1000 and resuspend the cells in one milliliter of plating medium. Perform a cell count using hemocytometer and then dilute the cell suspension to 1000 cells per microliter with plating medium.Plate 120 microliters of the cell suspension onto poly-D-lysine, poly-L-ornithine, and laminin coated culture dishes immediately after aspiration of laminin, to ensure that the coating does not dry and form an uneven surface. Then, after one hour of incubation, carefully add three milliliters of culture medium to an unseeded area of the culture dish to minimize disruption of cells. After three weeks of culture, rinse the culture dish with two milliliters RNase-free Dulbecco's PBS.Remove the DPBS. Then replace with one milliliter of fresh DPBS for harvesting. Use the GFP filter set and 40X objective on an inverted epifluoresence microscope to identify fluorescent TH positive neurons in the cultures.Use the micromanipulator to the pipette over the cell soma. Then use plastic tubing attached to the side port of the micropipette holder to apply gentle suction in order to aspirate the neuron into the glass micropipette. Immediately remove the micropipette containing the cell from the bathing solution and place the tip inside a 0.2 millilter PCR tube collection containing reaction buffer.Break the tip against the side of the tube near the bottom. Then place a sterile 21 gauge syringe needle in the back of the micorpipette and apply pressure to expel the remaining fluid from the broken tip of the micropipette. Centrifuge the collection tube for five seconds using a desktop microcentrifuge and then freeze it in dry ice.While working in the PCR cabinet with reagents on ice, or on a chiller block, prepare the first strain master mix plus 10%additional volume at room temperature. And one microliter of three prime primer one and one microliter of quantified RNA Spikes to the sample. Then incubate the sample in the thermocycler for three minutes at 72 degrees Celsius and then put the samples directly onto the PCR cooler rack.Next, add 5.5 microliters of the prepared master mix to the reaction tube in a PCR cooler rack and mix by pipetting gently. After centrifuging the tube for five seconds incubate in a thermocycler set to the parameters now shown on screen. To purify the first-strand cDNA add 25 microliters of vortexed room temperature magnetic beads to the sample.Mix by pipetting the entire volume up and down at least 10 times. Then incubate at room temperature for eight minutes to allow the cDNA to bind to the beads. Then place the samples and magnetic beads on the magnetic separation device until the liquid appears completely clear and there are no beads left in the supernatant.Aspirate and discard the supernatant. Spin the sample for five seconds to collect the liquid from the side of the tube. Place the samples on the magnetic separation device for 30 seconds.Then remove all the remaining supernatant with the P10 pipetter to leave just the beads with bound DNA. Add 50 microliters of the ds-cDNA PCR master mix and then run the second strand synthesis PCR program to obtain the double stranded cDNA. This histogram shows the number of protein coding genes detected in single cell dopaminergic RNA sequencing libraries generated from TH-eGFP positive cells in fragments per kilobase of transcript per million mapped reads.6, 000 to 8, 000 protein genes were detected in the single cell libraries. Once mastered this technique can be done in four to five weeks, including time to derive the cells, allow for the maturation of the cells in culture, the cell harvesting and the library generation steps, the sequencing of the libraries, and the preliminary analysis. While attempting this procedure, it's important to remember to keep an RNAse-free workspace for the RNA seek library generation and cell harvesting, to maintain sterile techniques through the cell culturing and to make pipettes with appropriately sized diameter for the single cell harvesting.Don't forget that working with paraformaldehyde can be extremely hazardous and precautions such as using a fume hood, avoiding skin and eye contact, keeping away from heat and open flames, and wearing the appropriate personal protective clothing should always be taken. Following this procedure, other methods like gene expression and gene network analysis can be performed in order to answer additional questions such as how drug treatments effect protein expression in dopaminergic cells.

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JoVE Journal

Neuroscience

9.3K Views.  California Institute of Technology (Caltech). The overall goal of this procedure is to reliably identify living dopaminergic neurons in midbrain cultures using tyrosine hydroxlase driven eGFP expression, single cell harvesting, and RNA sequencing library generation. This method can help answer key questions in the Parkinson's disease and drug addiction fields, because it makes it possible to perform gene expression and electrophysiological assays on identified living dopaminergic neurons in culture. The advantage of this technique is tha...