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

Summary

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.

Abstract

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.

Introduction

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 cells^1,2,3. 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....

Protocol

All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Northwestern University.

1. Preparation of Solutions for Electrophysiology (4 h)

1. Make 0.1% DEPC-treated H₂O by adding 1 mL of diethyl pyrocarbonate (DEPC) to 999 mL of reverse osmosis-purified H₂O. Mix thoroughly and let the mixture incubate for 1 h at room temperature (RT). Then, autoclave the DEPC-mixed H₂O for 15 min on a liquid cycle. Let the DEPC-treated H₂O cool at RT.
2. Make the extracellular solution by mixing one bottle of Ames' medium and 1.9 g (23 mM) of sodium bicarbonate ....

Results

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 .......

Discussion

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.......

Materials

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