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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 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.
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.
All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Northwestern University.
1. Preparation of Solutions for Electrophysiology (4 h)
2. Preparation of Retinal Tissue (2 h)
NOTE: All procedures in this section should be performed under dim red illumination
3. Visualization and Targeting of GFP+ Retinal Ganglion Cells (10 min)
NOTE: All procedures in this section should be performed under dim red illumination
4. Cell Isolation (2 min)
5. RNA Purification (30 min)
6. Reverse Transcription (10 min)
NOTE: Before beginning, thaw the necessary reagents for reverse transcription (RT; except for the enzyme) on ice. These include: primer II, buffer 1, oligonucleotide, and RNase inhibitor.
7. cDNA Amplification (2.5 h)
NOTE: Before beginning, thaw PCR buffer and PCR primer on ice and spin the tubes down in a tabletop mini centrifuge before making the PCR master mix.
8. Purification of Amplified cDNA (30 min)
9. Determine Concentrations and Tagment cDNA (20 min)
10. Index Coupling and Purification (1 h)
NOTE: Before beginning, bring the DNA beads and resuspension buffer to RT for at least 30 min. Decide which indices to use for each of the samples.
NOTE: These indices will be attached to the respective 5' and 3' ends of the fragmented DNA for the identification of samples following sequencing. Ensure that no two pairings are the same for samples that may be sequenced together. For example, if sample 1 will use indices white 1 and orange 1, sample two should use white 1 and orange 2 or white 2 and orange 2, but never the same combination of indices. This kit contains 4 distinct white and 6 distinct orange indices. All of the different possible combinations allow up to 24 samples to be pooled in one sequencing lane. Although we typically only pool 10 samples in a lane, one could also use the kit containing 24 indices, which would allow for the pooling of 96 samples in a single lane of sequencing, if desired.
11. Pooling of Samples (10 min)
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 ...
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...
The authors have nothing to disclose.
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.
Name | Company | Catalog Number | Comments |
Ames' Medium | Sigma Aldrich | A1420-10X1L | |
Sodium Bicarbonate | Sigma Aldrich | S8875 | |
K-gluconate | Spectrum Chemical | PO178 | |
EGTA | Sigma Aldrich | E4378 | |
HEPES | Sigma Aldrich | H3375 | |
Diethyl pyrocarbonate (DEPC) | Sigma Aldrich | D5758 | |
Alexa Fluor 594 Hydrazide | Invitrogen | A10442 | |
Collagenase | Worthington Biochemical | LS005273 | |
Hyaluronidase | Worthington Biochemical | LS002592 | |
Petri dish (35mm diameter) | Thermo Fisher Scientific | 153066 | |
Ophthalmologic scissors | Fine Science Tools | 15000-00 | |
#5 Forceps | Fine Science Tools | 11252-30 | |
Microplate Shaker | Fisher Scientific | 13-687-708 | |
Glass Micropipette | Sutter | BF120-69-10 | |
Micropipette Puller | Sutter | P-1000 horizontal pipette puller | |
1mL syringe | Fisher Scientific | 14-823-2F | |
Flexible tubing | Fisher Scientific | 14-171 | |
TCL lysis buffer | Qiagen | 1031576 | Lysis Buffer 1 |
β-mercaptoethanol | Sigma Aldrich | M3148 | |
RNase-Free Water | Qiagen | 129112 | |
0.2 ml PCR tubes | Eppendorf | 30124359 | |
Ethyl Alcohol, Pure | Sigma Aldrich | E7023 | Ethanol |
Analog Vortex Mixer | Thermo Fisher Scientific | 02215365 | Vortex |
Mini Centrifuge | Thermo Fisher Scientific | 05-090-100 | |
Agencourt RNAClean XP Beads | Beckman Coulter | A63987 | RNA magnetic beads |
MagnaBlot II Magnetic Separator | Promega | V8351 | Magnetic stand |
1.5 ml MCT Graduated Tubes | Thermo Fisher Scientific | 05-408-129 | |
Smart-Seq v4 Ultra Low Input RNA Kit | Clontech | 634888 | Reagents for Reverse Transcription and PCR Amplification |
10X Lysis Buffer | Lysis Buffer 2 | ||
5X Ultra Low First-Strand Buffer | Buffer 1 | ||
3' SMART-Seq CDS Primer II A | Primer II | ||
SMART-Seq v4 Oligonucleotide | Oligonucleotide | ||
SMARTScribe Rverse Transcriptase | Reverse Transcriptase | ||
2X SeqAmp PCR Buffer | PCR Buffer | ||
PCR Primer II A | PCR Primer | ||
SeqAmp DNA Polymerase | DNA Polymerase | ||
Mastercycler pro S | Eppendorf | 950030020 | Thermocycler |
Agencourt AMPure XP Beads | Beckman Coulter | A63881 | DNA magnetic beads |
2100 Bioanalyzer | Agilent Technologies | G2939AA | |
HS Bioanalyzer Chips & Reagents | Agilent Technologies | 5067-4626 | |
Qubit HS Assay Kit | Thermo Fisher Scientific | Q32851 | For the calculation of sample concentrations |
Qubit Assay Tubes | Thermo Fisher Scientific | Q32856 | |
Qubit 2.0 Fluorometer | Thermo Fisher Scientific | Q32866 | |
Nextera XT DNA Sample Preparation Kit | Illumina | FC-131-1024 | Reagents for Tagmentation and Index Coupling |
TD Buffer | Buffer 2 | ||
ATM | Tagmentation Mix | ||
NT Buffer | Tagmentation Neutralizing Buffer | ||
NPM | PCR Master Mix | ||
Nextera XT Index Kit | Illumina | FC-131-1001 | Indices for Tagmentation |
N501 | White 1 | ||
N502 | White 2 | ||
N701 | Orange 1 | ||
N702 | Orange 2 | ||
HiSeq 2500 | Illumina | SY-401-2501 | For completing sequencing of samples |
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