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This protocol describes the manual sorting procedure to isolate single fluorescently labeled neurons followed by in vitro transcription-based mRNA amplification for high-depth single-cell RNA sequencing.
Single-cell RNA sequencing (RNA-seq) is now a widely implemented tool for assaying gene expression. Commercially available single-cell RNA-sequencing platforms process all input cells indiscriminately. Sometimes, fluorescence-activated cell sorting (FACS) is used upstream to isolate a specifically labeled population of interest. A limitation of FACS is the need for high numbers of input cells with significantly labeled fractions, which is impractical for collecting and profiling rare or sparsely labeled neuron populations from the mouse brain. Here, we describe a method for manually collecting sparse fluorescently labeled single neurons from freshly dissociated mouse brain tissue. This process allows for capturing single-labeled neurons with high purity and subsequent integration with an in vitro transcription-based amplification protocol that preserves endogenous transcript ratios. We describe a double linear amplification method that uses unique molecule identifiers (UMIs) to generate individual mRNA counts. Two rounds of amplification results in a high degree of gene detection per single cell.
Single-cell RNA sequencing (RNA-seq) has transformed transcriptomic studies. While large-scale single-cell RNAseq can be performed using a variety of techniques, such as droplets1,2, microfluidics3, nanogrids4, and microwells5, most methods cannot sort defined cell types that express genetically encoded fluorophores. To isolate a select cell population, fluorescence-activated cell sorting (FACS) is often used to sort labeled cells in a single-cell mode. However, FACS has some restrictions and requires meticulous sample processing steps. First, a large number of input cells are typically needed (often several million cells per mL), with a significant fraction (>15–20%) containing the labeled population. Second, cell preparations may require multiple rounds of density gradient centrifugation steps to remove glial fraction, debris, and cell clumps that might otherwise clog the nozzle or flow cell. Third, FACS usually employs staining and destaining steps for live/dead staining (e.g., 4′,6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), and Cytotracker dyes), which take up additional time. Fourth, as a rule of thumb for two-color sorting (such as DAPI and green/red fluorescent protein (GFP/RFP)), usually two samples and one control are needed, requiring an unlabeled sample to be processed in addition to the desired mouse strain. Fifth, filtering is often performed multiple times before and during sample sorting to proactively prevent clogged sample lines in an FACS machine. Sixth, time must be allotted in most commonly used FACS setups to initialize and stabilize the fluid stream and perform droplet calibration. Seventh, control samples are typically run in sequence prior to the actual sample collection to set up compensation matrices, doublet rejection, setting gates, etc. Users either perform steps six and seven themselves ahead of time or require the assistance of a technician in parallel. Finally, post-FACS, there are often steps to ensure that only labeled single cells are present in each well; for example, by checking samples in a high-content screening setup such as a fast plate imager.
To circumvent the steps outlined above and facilitate a relatively quick, targeted sequencing of a small population of single fluorescently labeled neurons, we describe a manual sorting procedure followed by two rounds of a highly sensitive in vitro amplification protocol, called double in-vitro transcription with absolute counts sequencing (DIVA-Seq). The RNA amplification and cDNA library generation are adapted from Eberwine et al.6 and Hashimshony et al.7, with certain modifications to suit mouse interneurons that have smaller cellular volumes; furthermore, we have also found that it is equally useful for excitatory pyramidal neurons.
All the procedures including animal subjects have been approved by IACUC at Cold Spring Harbor Laboratory, NY (IACUC #16-13-09-8).
1. Manual Sorting of Fluorescently Labeled Mouse Neurons
2. First Round RNA Amplification
NOTE: The following procedure is for single strip of eight 0.2 mL microfuge tubes. Scale the reactions as needed.
3. Second Round Amplification
4. Amplified RNA Fragmentation and Cleanup
5. Library Preparation
NOTE: IVTs can be pooled at this point, if there is no overlap in barcodes used. The phosphatase treatment time is 40 min. Poly-nucleotide kinase treatment time is 1 h.
6. PCR Product Cleanup and Size Selection
7. Determination of Library Amount and Quality
8. Sample Submission
Using the protocol described above, GABAergic neurons were manually sorted (Figure 1) and RNA was amplified, then made into a cDNA library (Figure 2) and sequenced at high depth8. The amplified RNA (aRNA) products ranged between 200–4,000 bp in size, with a peak distribution slightly above 500 bp (Figure 3A). The bead-purified cDNA library was further size-restricted by ...
The manual sorting protocol is suitable for a supervised RNA sequencing of neuron populations that are either sparsely labeled in the mice brain or are representing a rare cell population that is otherwise not feasible to study using current high-throughput cell sorting and amplification methods. Cells subjected to FACS usually undergo sheath and sample line pressures in the range of ~9–14 psi, depending on nozzle size and desired event rates. In addition, upon being ejected from the nozzle, the cells can land hard...
Authors declare that there are no competing financial interests.
This work was supported by grants from the NIH (5R01MH094705-04 and R01MH109665-01 to Z.J.H.), by the CSHL Robertson Neuroscience Fund (to Z.J.H.), and by a NARSAD Post-Doctoral Fellowship (to A.P.).
Name | Company | Catalog Number | Comments |
ERCC RNA Spike-In Control Mixes | Thermo Fisher | Cat# 4456740 | |
SuperScript III | Thermo Fisher | Cat# 18080093 | |
RNaseOUT Recombinant Ribonuclease Inhibitor | Thermo Fisher | Cat# 10777019 | |
RNA fragmentation buffer | New England Biolabs | Cat# E6105S | |
RNA MinElute kit | Qiagen | Cat# 74204 | |
Antarctic phosphatase | New England Biolabs | Cat# M0289 | |
Poly nucleotide kinase | New England Biolabs | Cat# M0201 | |
T4 RNA ligase2, truncated | New England Biolabs | Cat# M0242 | |
Ampure Xp magnetic beads | Beckman Coulter | Cat# A63880 | |
SPRIselect size selection magnetic beads | Thermo Fisher | Cat# B23317 | |
DL-AP5 | Tocris | Cat# 0105 | |
CNQX | Tocris | Cat# 1045 | |
TTX | Tocris | Cat# 1078 | |
Protease from Streptomyces griseus | Sigma-Aldrich | Cat# P5147 | |
Message Amp II kit | Thermo Fisher | Cat# AM1751 | |
Carbogen | Airgas | Cat# UN3156 | |
Sylgard 184 | Sigma-Aldrich | Cat# 761036 | |
Illumina TrueSeq smallRNA kit | Illumina | Cat# RS-200-0012 | |
Bioanalyzer RNA Pico chip | Agilent | Cat# 5067-1513 | |
Bioanalyzer High Sensitvity DNA chip | Agilent | Cat# 5067-4626 | |
Bioanalyzer 2100 | Agilent | ||
Dissection microscope with fluorescence and bright field illumination with DIC optics. (Leica model MZ-16F). | Leica | Model MZ-16F | |
Glass microcapillary: Borosilicate capillary tubes 500/pk. OD= 1 mm, ID=0.58 mm, wall= 0.21 mm, Length= 150 mm. | Warner instruments | Model GC100-15, Order# 30-0017 | |
Capillary pipette puller | Sutter Instruments Co | P-97 | |
Vibratome | Thermo Microm | HM 650V | |
Vibratome tissue cooling unit | Thermo Microm | CU 65 |
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