We would like to acknowledge Jennifer Bair and Einat Snir, as well as the University of Iowa Institute for Human Genetics, for their assistance in preparing and handling samples.

<ol>
	<li>Austin, C., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+Cell+Fate+in+the+Vertebrate+Retina.">Specification of Cell Fate in the Vertebrate Retina.</a> Neural Cell Specif. 3, 139-143 (1995).</li><li>Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Neuronal+Organization+of+the+Retina.">The Neuronal Organization of the Retina.</a> Neuron. 76 (2), 266-280 (2012).</li><li>Rodieck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The First Steps in Seeing. , (1998).</li><li>Chiu, I. 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+profiling+at+whole+population+and+single+cell+levels+reveals+somatosensory+neuron+molecular+diversity.">Transcriptional profiling at whole population and single cell levels reveals somatosensory neuron molecular diversity.</a> Elife. 3, e04660 (2014).</li><li>Tasic, 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=Adult+mouse+cortical+cell+taxonomy+revealed+by+single+cell+transcriptomics.">Adult mouse cortical cell taxonomy revealed by single cell transcriptomics.</a> Nat. Neurosci. 19 (2), 335-346 (2016).</li><li>Trapnell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+cell+types+and+states+with+single-cell+genomics.">Defining cell types and states with single-cell genomics.</a> Genome Res. 25 (10), 1491-1498 (2015).</li><li>Zeisel, 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=Cell+types+in+the+mouse+cortex+and+hippocampus+revealed+by+single-cell+RNA-seq.">Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq.</a> Science. 347 (6226), 1138-1142 (2015).</li><li>Schmidt, T. M., Do, M. T. H., Dacey, D., Lucas, R., Hattar, S., Matynia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanopsin-Positive+Intrinsically+Photosensitive+Retinal+Ganglion+Cells:+From+Form+to+Function.">Melanopsin-Positive Intrinsically Photosensitive Retinal Ganglion Cells: From Form to Function.</a> J. Neurosci. 31 (45), 16094-16101 (2011).</li><li>Eberwine, J., Miyashiro, K., Kacharmina, J. E., Job, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+translation+of+classes+of+mRNAs+that+are+targeted+to+neuronal+dendrites.">Local translation of classes of mRNAs that are targeted to neuronal dendrites.</a> Proc. Natl. Acad. Sci. 98 (13), 7080-7085 (2001).</li><li>Darmanis, 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+survey+of+human+brain+transcriptome+diversity+at+the+single+cell+level.">A survey of human brain transcriptome diversity at the single cell level.</a> Proc. Natl. Acad. Sci. 112 (23), 7285-7290 (2015).</li><li>Macosko, E. 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=Highly+Parallel+Genome-wide+Expression+Profiling+of+Individual+Cells+Using+Nanoliter+Droplets.">Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets.</a> Cell. 161 (5), 1202-1214 (2015).</li><li>Usoskin, 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=Unbiased+classification+of+sensory+neuron+types+by+large-scale+single-cell+RNA+sequencing.">Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing.</a> Nat. Neurosci. 18 (1), 145-153 (2015).</li><li>Poulin, J. F., Zou, J., Drouin-Ouellet, J., Kim, K. Y. A., Cicchetti, F., Awatramani, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+midbrain+dopaminergic+neuron+diversity+by+single-cell+gene+expression+profiling.">Defining midbrain dopaminergic neuron diversity by single-cell gene expression profiling.</a> Cell. Rep. 9 (3), 930-943 (2014).</li><li>Gross, A., Schoendube, J., Zimmermann, S., Steeb, M., Zengerle, R., Koltay, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technologies+for+Single-Cell+Isolation.">Technologies for Single-Cell Isolation.</a> Int. J. Mol. Sci. 16, 16897-16919 (2015).</li><li>Lovatt, 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=Transcriptome+in+vivo+analysis+(TIVA)+of+spatially+defined+single+cells+in+live+tissue.">Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue.</a> Nat Methods. 11 (2), 190-196 (2014).</li><li>Qiu, 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=Single-neuron+RNA-Seq:+Technical+feasibility+and+reproducibility.">Single-neuron RNA-Seq: Technical feasibility and reproducibility.</a> Front. Genet. 3 (124), 1-8 (2012).</li><li>Subkhankulova, T., Yano, K., Robinson, H. P. C., Livesey, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grouping+and+classifying+electrophysiologically-defined+classes+of+neocortical+neurons+by+single+cell,+whole-genome+expression+profiling.">Grouping and classifying electrophysiologically-defined classes of neocortical neurons by single cell, whole-genome expression profiling.</a> Front. Mol. Neurosci. 3 (10), 1-11 (2010).</li><li>Fuzik, 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=Integration+of+electrophysiological+recordings+with+single-cell+RNA-seq+data+identifies+neuronal+subtypes.">Integration of electrophysiological recordings with single-cell RNA-seq data identifies neuronal subtypes.</a> Nat. Biotechnol. 34 (2), 175-183 (2016).</li><li>Schmidt, T. M., Kofuji, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+isolated+retinal+preparation+to+record+light+response+from+genetically+labeled+retinal+ganglion+cells.">An isolated retinal preparation to record light response from genetically labeled retinal ganglion cells.</a> J. Vis. Exp. (47), e2367 (2011).</li><li>Schmidt, T. M., Kofuji, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+morphological+differences+among+intrinsically+photosensitive+retinal+ganglion+cells.">Functional and morphological differences among intrinsically photosensitive retinal ganglion cells.</a> J Neurosci. 29 (2), 476-482 (2009).</li><li>Schmidt, T. M., Kofuji, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+Function+of+Bistratified+Intrinsically+Photosensitive+Retinal+Ganglion+Cells+in+the+Mouse.">Structure and Function of Bistratified Intrinsically Photosensitive Retinal Ganglion Cells in the Mouse.</a> J Comp Neurol. 519 (8), 1492-1504 (2011).</li><li>Schmidt, T. M., Kofuji, 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+cone+pathway+influence+on+intrinsically+photosensitive+retinal+ganglion+cell+subtypes.">Differential cone pathway influence on intrinsically photosensitive retinal ganglion cell subtypes.</a> J Neurosci. 30 (48), 16262-16271 (2010).</li><li>Clontech Laboratories I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> SMARTerTM Ultra Low RNA Kit for Illumina&#174; Sequencing. , (2013).</li><li>Trombetta, J. J., Gennert, D., Lu, D., Satija, R., Shalek, A. K., Regev, 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+of+single-cell+RNA-Seq+libraries+for+next+generation+sequencing.">Preparation of single-cell RNA-Seq libraries for next generation sequencing.</a> Curr. Protoc. Mol. Biol. 4 (22), 1-17 (2014).</li><li>Ecker, J. L., 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=Melanopsin-expressing+retinal+ganglion-cell+photoreceptors:+Cellular+diversity+and+role+in+pattern+vision.">Melanopsin-expressing retinal ganglion-cell photoreceptors: Cellular diversity and role in pattern vision.</a> Neuron. 67 (1), 49-60 (2010).</li><li>Schmidt, T. 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=A+Role+for+Melanopsin+in+Alpha+Retinal+Ganglion+Cells+and+Contrast+Detection.">A Role for Melanopsin in Alpha Retinal Ganglion Cells and Contrast Detection.</a> Neuron. 82 (4), 781-788 (2014).</li><li>Sanes, J. R., Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Types+of+Retinal+Ganglion+Cells:+Current+Status+and+Implications+for+Neuronal+Classification.">The Types of Retinal Ganglion Cells: Current Status and Implications for Neuronal Classification.</a> Annu. Rev. Neurosci. 38, 221-246 (2014).</li><li>Goetz, J. J., Trimarchi, J. M. <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+Profiling+of+Developing+and+Mature+Retinal+Neurons.">Single-cell Profiling of Developing and Mature Retinal Neurons.</a> J. Vis. Exp. , e3824 (2012).</li><li>Trimarchi, J. 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=Molecular+Heterogeneity+of+Developing+Retinal+Ganglion+and+Amacrine+Cells+Revealed+through+Single+Cell+Gene+Expression+Profiling.">Molecular Heterogeneity of Developing Retinal Ganglion and Amacrine Cells Revealed through Single Cell Gene Expression Profiling.</a> J. Comp. Neurol. 502, 1047-1065 (2007).</li></ol>

The authors have nothing to disclose.

Our protocol demonstrates, through a quick and easy-to-use guide, a method to prepare single cells of identified morphological classes for high-quality sequencing, with little injury to the sample. In the present manuscript, intrinsically photosensitive retinal ganglion cells are morphologically characterized, isolated, and prepared for RNA-Seq. Cellular stresses may occur during retinal handling; for this reason, we replace each piece of tissue after no more than 4 h of use. We can assess the state of the cells by using...

Neuronal diversity is observed throughout the central nervous system, particularly in the vertebrate retina, a highly specialized tissue consisting of 1 glial and 6 neuronal cell types that arise from one population of retinal progenitor cells1,2,3. Many subtypes of cells can be classified functionally, morphologically, and genetically. The goal of this protocol is to tie the genetic variability of cell types to their identifiable functional and/or morphological characteristics. A number of genes have been identified for the classification of cells, but many subtypes continue to go uncharacterized, as they represent a small fraction of the overall population. The identification of genes within these specific subtypes will allow for a greater understanding of neuronal diversity within the retina and may also shed light on the diversification of neural cells elsewhere. Furthermore, single-cell studies allow for the uncovering of new cell types, which may have been overlooked due to their low representation among the overall population4,5,6,7.
One of the benefits of single-cell transcriptomics is that unique markers or combinations of markers that define a particular cellular subtype can be discovered. These can then be used to gain genetic access to that cell type for different manipulations. For example, we are using this protocol to characterize the cell type-specific genes of a subset of retinal ganglion cells that express the photopigment melanopsin. The use of a fluorescent marker in melanopsin-expressing retinal ganglion cells enables the study of these cells, as they are clustered together due to their expression of a known gene. Interestingly, there are five known subtypes of this cell population in the mouse retina8. Thus, in order to isolate RNA from cells of each type, we have used established morphological classifications within the transgenic model to identify each subtype prior to cell isolation. This technique allows for the characterization of cells as well as for their isolation directly from the retina, without the need for tissue dissociation, which may cause a stress response within cells and contamination due to severed dendrites9.
A multitude of new techniques have come to light in the past few years as the RNA-Seq method continues to develop. These tools allow for maximized cell acquisition and greater cost efficiency while approaching the question at hand4,7,10,11,12,13. However, while these techniques have been excellent stepping stones, there are a number of hurdles still encountered that this protocol is able to address. First, many of the current procedures isolate cells from dissociated tissue and attempt to use either principal component analysis or hierarchical clustering post-hoc to determine cell classification. Relying on these tools to classify subtypes may not produce reliable results and may force one to find new ways to validate this data for the correlation of a genetic marker to a functional cell type. The requirement for dissociation in other protocols can sometimes result in tissue damage and can cause neuronal processes to be severed, resulting in a potential loss of mRNA. Furthermore, in dissociated cell preparations, the stress responses may begin to affect the transcriptomes of these cells14. This protocol overcomes these challenges by determining the functional cell type prior to isolation, and it better maintains the health of the cells by keeping the retinal tissue intact.
One technique was introduced in 2014 and consisted of the in vivo analysis of the transcriptome of live cells15. While this technique allows for the examination of the transcriptome with minimal mechanical disruption to the tissue, it lacks the ability to classify specific cell types within the tissue before examining their transcriptomes without using a very specific reporter mouse. Our protocol does not require a specific reporter, as we utilize cell filling and electrophysiology to characterize cells before their isolation. Another limitation of this previous protocol is that it requires a specific wavelength to excite the photoactivatable element, whereas our protocol allows for the use of a fluorescent reporter and fluorescent dye, which are readily available or can be chosen by each lab individually. Still, other laboratories have married the two methods of electrophysiology and transcriptomics for the study of cellular diversity. The use of patch-clamp recordings to characterize the function of a cell prior to its isolation has been performed on dissociated neurons16 and, in some cases, it has preceded the use of microarray analysis17 for these studies. The same complications are encountered by those approaches, as they require tissue dissociation or the use of microarray technology, which relies on the hybridization of samples to available probes. One of the most recent advances has been the development of Patch-Seq, a technique that combines the use of patch-clamp recordings and RNA-Seq technology to understand cells from whole-brain slices18. While this technique has its similarities to the protocol presented here, it is again important to note that our approach allows the tissue to remain intact for the health of the cells. Here, we present a protocol for the optimization of this alliance, which generates high-quality, single-cell libraries for the use of RNA-Seq to obtain a high read depth and mapping coverage.

<table><tbody><tr><td>Ames&#039; Medium</td><td>Sigma Aldrich</td><td>A1420-10X1L</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma Aldrich</td><td>S8875</td><td></td></tr><tr><td>K-gluconate</td><td>Spectrum Chemical</td><td>PO178</td><td></td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E4378</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H3375</td><td></td></tr><tr><td>Diethyl pyrocarbonate (DEPC)</td><td>Sigma Aldrich</td><td>D5758</td><td></td></tr><tr><td>Alexa Fluor 594 Hydrazide</td><td>Invitrogen</td><td>A10442</td><td></td></tr><tr><td>Collagenase</td><td>Worthington Biochemical&amp;nbsp;</td><td>LS005273</td><td></td></tr><tr><td>Hyaluronidase</td><td>Worthington Biochemical&amp;nbsp;</td><td>LS002592</td><td></td></tr><tr><td>Petri dish (35 mm diameter)</td><td>Thermo Fisher Scientific</td><td>153066</td><td></td></tr><tr><td>Ophthalmologic scissors</td><td>Fine Science Tools</td><td>15000-00</td><td></td></tr><tr><td>#5 Forceps</td><td>Fine Science Tools</td><td>11252-30</td><td></td></tr><tr><td>Microplate Shaker</td><td>Fisher Scientific</td><td>13-687-708</td><td></td></tr><tr><td>Glass Micropipette</td><td>Sutter</td><td>BF120-69-10</td><td></td></tr><tr><td>Micropipette Puller</td><td>Sutter</td><td></td><td>P-1000 horizontal pipette puller</td></tr><tr><td>1 mL syringe</td><td>Fisher Scientific</td><td>14-823-2F</td><td></td></tr><tr><td>Flexible tubing</td><td>Fisher Scientific</td><td>14-171</td><td></td></tr><tr><td>TCL lysis buffer</td><td>Qiagen</td><td>1031576</td><td>Lysis Buffer 1</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma Aldrich</td><td>M3148</td><td></td></tr><tr><td>RNase-free Water</td><td>Qiagen</td><td>129112</td><td></td></tr><tr><td>0.2 mL PCR tubes</td><td>Eppendorf</td><td>30124359</td><td></td></tr><tr><td>Ethyl Alcohol, Pure</td><td>Sigma Aldrich</td><td>E7023</td><td>Ethanol</td></tr><tr><td>Analog Vortex Mixer</td><td>Thermo Fisher Scientific</td><td>02215365</td><td>Vortex</td></tr><tr><td>Mini Centrifuge</td><td>Thermo Fisher Scientific</td><td>05-090-100</td><td></td></tr><tr><td>Agencourt RNAClean XP Beads</td><td>Beckman Coulter</td><td>A63987</td><td>RNA magnetic beads</td></tr><tr><td>MagnaBlot II Magnetic Separator</td><td>Promega</td><td>V8351</td><td>Magnetic stand</td></tr><tr><td>1.5 mL MCT Graduated Tubes</td><td>Thermo Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Smart-Seq v4 Ultra Low Input RNA Kit</td><td>Clontech</td><td>634888</td><td>Reagents for Reverse Transcription and PCR Amplification</td></tr><tr><td>10x Lysis Buffer</td><td></td><td></td><td>Lysis Buffer 2</td></tr><tr><td>5x Ultra Low First-strand Buffer</td><td></td><td></td><td>Buffer 1</td></tr><tr><td>3&#039; SMART-Seq CDS Primer II A</td><td></td><td></td><td>Primer II</td></tr><tr><td>SMART-Seq v4 Oligonucleotide</td><td></td><td></td><td>Oligonucleotide</td></tr><tr><td>SMARTScribe Rverse Transcriptase</td><td></td><td></td><td>Reverse Transcriptase (RT)</td></tr><tr><td>2x SeqAmp PCR Buffer</td><td></td><td></td><td>PCR Buffer</td></tr><tr><td>PCR Primer II A</td><td></td><td></td><td>PCR Primer</td></tr><tr><td>SeqAmp DNA Polymerase</td><td></td><td></td><td>DNA Polymerase</td></tr><tr><td>Mastercycler pro S</td><td>Eppendorf</td><td>950030020</td><td>Thermocycler</td></tr><tr><td>Agencourt AMPure XP Beads</td><td>Beckman Coulter</td><td>A63881</td><td>DNA magnetic beads</td></tr><tr><td>2100 Bioanalyzer</td><td>Agilent Technologies</td><td>G2939AA</td><td></td></tr><tr><td>HS Bioanalyzer Chips &amp;amp; Reagents</td><td>Agilent Technologies</td><td>5067-4626</td><td></td></tr><tr><td>Qubit HS Assay Kit</td><td>Thermo Fisher Scientific</td><td>Q32851</td><td>For the calculation of sample concentrations</td></tr><tr><td>Qubit Assay Tubes</td><td>Thermo Fisher Scientific</td><td>Q32856</td><td></td></tr><tr><td>Qubit 2.0 Fluorometer</td><td>Thermo Fisher Scientific</td><td>Q32866</td><td></td></tr><tr><td>Nextera XT DNA Sample Preparation Kit</td><td>Illumina</td><td>FC-131-1024</td><td>Reagents for Tagmentation and Index Coupling</td></tr><tr><td>TD Buffer</td><td></td><td></td><td>Buffer 2</td></tr><tr><td>ATM</td><td></td><td></td><td>Tagmentation Mix</td></tr><tr><td>NT Buffer</td><td></td><td></td><td>Tagmentation Neutralizing Buffer</td></tr><tr><td>NPM</td><td></td><td></td><td>PCR Master Mix</td></tr><tr><td>Nextera XT Index Kit</td><td>Illumina</td><td>FC-131-1001</td><td>Indices for Tagmentation</td></tr><tr><td>N501</td><td></td><td></td><td>White 1</td></tr><tr><td>N502</td><td></td><td></td><td>White 2</td></tr><tr><td>N701</td><td></td><td></td><td>Orange 1</td></tr><tr><td>N702</td><td></td><td></td><td>Orange 2</td></tr><tr><td>HiSeq 2500</td><td>Illumina</td><td>SY-401-2501</td><td>For completing sequencing of samples</td></tr></tbody></table>

single-cell rna-seq of defined subsets of retinal ganglion cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Cell types are easily classified following the dye injection
Figure 1 shows an example of a GFP+ RGC before and after fluorescent tracer filling. This cell was identified based on its expression of GFP in the transgenic line (Figure 1A). A tight seal was formed with a fine-tip, pulled-glass electrode onto the soma of this cell. In order to characterize the subtype, the fluorescent dye was injected ...

Watch this Scientific Journal Video about Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells at JoVE.com

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

single-cell-rna-seq-of-defined-subsets-of-retinal-ganglion-cells

Research

JoVE Journal

Neuroscience

13.7K Views.  Iowa State University. Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

Video: Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Bioengineering

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies ...

Developmental Biology

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during development and in disease states. Single-cell RNA sequencing allows us to comprehensively profile gene expression changes observed in retinal development and disease at a cellular level, while single-cell ATAC-Seq allows analysis of chromatin accessibility and transcription factor binding to be profiled at similar resolution. Here the use of these techniques in the developing retina is described, and MULTI-Seq is demonstrated, where individual samples are labeled with a modified oligonucleotide-lipid complex, enabling researchers to both increase the scope of individual experiments and substantially reduce costs.

Here, the authors showcase the utility of MULTI-seq for phenotyping and subsequent paired scRNA-seq and scATAC-seq in characterizing the transcriptomic and chromatin accessibility profiles in retina.

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during ...

Longitudinal In Vivo Imaging of Retinal Cells with Tailored Adeno-Associated Virus Serotypes via Confocal Scanning Laser Ophthalmoscopy

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding and treatment of ocular diseases. This study introduces an optimized adeno-associated virus (AAV) approach, utilizing specific serotypes and promoters to achieve optimal transfection efficiency in targeted retinal cells, including retinal ganglion cells (RGCs) and M&#252;ller glia. Leveraging the precision of confocal scanning laser ophthalmoscopy (CSLO), this work presents a non-invasive method for in vivo imaging that captures the longitudinal expression of AAV-mediated green fluorescent protein (GFP). This approach eliminates the need for terminal procedures, preserving the continuity of observation and the well-being of the subject. Furthermore, the GFP signal can be traced in AAV-infected RGCs along the visual pathway to the superior colliculus (SC) and lateral geniculate nucleus (LGN), enabling the potential for direct visual pathway mapping. These findings provide a detailed protocol and demonstrate the application of this powerful tool for real-time studies of retinal cell behavior, disease pathogenesis, and the efficacy of gene therapy interventions, offering valuable insights into the living retina and its connections.

This protocol describes the use of adeno-associated virus (AAV) vectors for cell-specific labeling and in vivo imaging using a confocal scanning laser ophthalmoscope (CSLO). This method enables the investigation of different retinal cell types and their contributions to retinal function and disease.

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding ...

Isolating Murine Retinal Ganglion Cells Using Fluorescence-Activated Cell Sorting

Source: Chintalapudi, S. R., et al. <a href="https://app.jove.com/v/55785">Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry.</a> J. Vis. Exp. (2017)
This video demonstrates the procedure for isolating murine primary retinal ganglion cells (RGCs) by using flow cytometry. This method allows for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparation of ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Quantification of Retinal Ganglion Cell Somas Following Cauterization-Induced Ocular Hypertension 

<a href='https://app.jove.com/v/63442/full-circle-cauterization-limbal-vascular-plexus-for-surgically'>Source: Lani-Louzada, R., et al.&#160; Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (2022).</a>This video demonstrates the quantification of retinal ganglion cells from chemically fixed rat retinas subjected to full-circle cauterization of the limbal vascular plexus. The process includes permeabilization, antibody staining, and nuclear counterstaining, followed by visualization under an epifluorescence microscope to assess the impact of elevated intraocular pressure on retinal ganglion cell viability.

Source: Lani-Louzada, R., et al.&#160; Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

Summary

Explore More Videos

Chapters in this video

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Single-cell Profiling of Developing and Mature Retinal Neurons

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq

Longitudinal In Vivo Imaging of Retinal Cells with Tailored Adeno-Associated Virus Serotypes via Confocal Scanning Laser Ophthalmoscopy

Isolating Murine Retinal Ganglion Cells Using Fluorescence-Activated Cell Sorting

Quantification of Retinal Ganglion Cell Somas Following Cauterization-Induced Ocular Hypertension