Name: single-cell rna sequencing of fluorescently labeled mouse neurons using manual sorting and double in vitro transcription with absolute counts sequencing (diva-seq)
Uploaded: 2018-10-26T15:00:00
Description: Watch this Scientific Journal Video about Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing DIVA-

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.).

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
<ol>
	<li>Pull glass microcapillaries (see Table of Materials) to 10&#8211;15 &#181;m exit diameter using a capillary puller with the following settings: heat: 508, pull: blank, vel: blank, time: blank.</li>
	<li>Attach 120&#8211;150 cm flexible silicone tubing (~0.8 mm inside diameter) to ...

<ol>
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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&#8211;14 psi, depending on nozzle size and desired event rates. In addition, upon being ejected from the nozzle, the cells can land hard...

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 (&#62;15&#8211;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&#8242;,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.

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			<td height="40" style="height:40px;width:261px;">ERCC RNA Spike-In Control Mixes</td>
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			<td height="40" style="height:40px;width:261px;">SuperScript III</td>
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			<td height="40" style="height:40px;width:261px;">RNaseOUT Recombinant Ribonuclease Inhibitor</td>
			<td>Thermo Fisher</td>
			<td>Cat# 10777019</td>
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			<td height="40" style="height:40px;width:261px;">RNA fragmentation buffer</td>
			<td>New England Biolabs</td>
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			<td>New England Biolabs</td>
			<td>Cat# M0289</td>
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			<td height="40" style="height:40px;width:261px;">Poly nucleotide kinase</td>
			<td>New England Biolabs</td>
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			<td height="40" style="height:40px;width:261px;">T4 RNA ligase2, truncated</td>
			<td>New England Biolabs</td>
			<td>Cat# M0242</td>
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			<td height="40" style="height:40px;width:261px;">Ampure Xp magnetic&#160; beads</td>
			<td>Beckman Coulter</td>
			<td>Cat# A63880</td>
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			<td height="40" style="height:40px;width:261px;">SPRIselect size selection magnetic beads</td>
			<td>Thermo Fisher</td>
			<td>Cat# B23317</td>
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			<td height="40" style="height:40px;width:261px;">DL-AP5</td>
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			<td height="40" style="height:40px;width:261px;">CNQX</td>
			<td>Tocris</td>
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			<td height="40" style="height:40px;width:261px;">Protease from Streptomyces griseus</td>
			<td>Sigma-Aldrich</td>
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			<td>Thermo Fisher</td>
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			<td>Sigma-Aldrich</td>
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			<td height="40" style="height:40px;width:261px;">Illumina TrueSeq smallRNA kit</td>
			<td>Illumina</td>
			<td>Cat# RS-200-0012</td>
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			<td>Agilent</td>
			<td>Cat# 5067-1513</td>
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			<td height="40" style="height:40px;width:261px;">Bioanalyzer High Sensitvity DNA chip</td>
			<td>Agilent</td>
			<td>Cat# 5067-4626</td>
			<td></td>
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			<td height="40" style="height:40px;width:261px;">Bioanalyzer 2100</td>
			<td>Agilent</td>
			<td></td>
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			<td height="84" style="height:84px;width:261px;">Dissection microscope with fluorescence and bright field illumination with DIC optics.&#160; (Leica model MZ-16F).</td>
			<td>Leica</td>
			<td>Model MZ-16F</td>
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			<td height="84" style="height:84px;width:261px;">Glass microcapillary: Borosilicate capillary tubes 500/pk. OD= 1 mm, ID=0.58 mm, wall= 0.21 mm, Length= 150 mm.</td>
			<td>Warner instruments</td>
			<td>Model GC100-15, Order# 30-0017</td>
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			<td height="21" style="height:21px;width:261px;">Capillary pipette puller</td>
			<td>Sutter Instruments Co</td>
			<td>P-97</td>
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			<td height="21" style="height:21px;width:261px;">Vibratome</td>
			<td style="width:123px;">Thermo Microm</td>
			<td>HM 650V</td>
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			<td height="21" style="height:21px;">Vibratome tissue cooling unit</td>
			<td>Thermo Microm</td>
			<td>CU 65</td>
			<td></td>
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single-cell rna sequencing of fluorescently labeled mouse neurons using manual sorting and double in vitro transcription with absolute counts sequencing (diva-seq)

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.

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&#8211;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 ...

Watch this Scientific Journal Video about Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing DIVA-

Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing DIVA-Seq

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 of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing (DIVA-Seq)

Single-cell RNA sequencing (RNA-seq) is now a widely implemented tool for assaying gene expression. Commercially available single-cell RNA-sequencing ...

This method can help solve a key obstacle in the field of single-neuron isolation and sequencing. Such as collecting specific population or a subpopulation of fluorescently labeled neuron from user-defined brain area. The main advantage of this technique is that it ensures the single neuron of interest is captured without contaminating debris.It is also relatively gentle, which is important for fragile neuron types that die quickly when subjected to the fluid pressures of conventional FACS sorting. Start by preparing pulled glass microcapillaries with 10 to 15 micron exit diameter using a capillary puller. Use fine scissors to cut the tip of the capillary to get the desired board diameter.Then use tubing connectors to attach a 120 to 150-centimeter length of flexible silicone tubing with 0.8 millimeter inside diameter to a 0.2 micron PVDF membrane syringe filter and a two-way tubing valve. Prepare 500 milliliters of chilled artificial cerebral spinal fluid or ACSF and oxygenate by bubbling 5%carbon dioxide balanced oxygen through an Airstone for 15 minutes or until the solution clears completely. Prepare three 150-milliliter beakers each containing 100 milliliters of ACSF.To one, add a cocktail of activity blockers. To another, add Streptococcus fraction for protease to a final concentration of one milligram per milliliter. To the final beaker, add FBS to a final concentration of 1%and oxygenate with 5%carbon dioxide-balanced oxygen through an Airstone.Finally, make three long-stemmed Pasteur pipettes with decreasing exit diameters by rolling them over an open flame. Dissect the brain from a euthanized mouse by opening the cranium with a small scissor and extracting the fresh brain using fine forceps without damaging the cortex. Place the brain in chilled and oxygenated ACSF leaving an Airstone attached to 5%carbon dioxide-balanced oxygen during the entire duration of the sectioning.Position the brain on the vibratome chuck and cut coronal or sagittal sections at 300 micron thickness. Collect as many sections as needed to obtain a minimum of 10 to 50 labeled cells. Put slices on a cotton-meshed slice holder placed inside a beaker so they are bathed in oxygenated ACSF.Move the slices into the beaker containing ACSF with activity blockers and block for 15 to 20 minutes at room temperature while bubbling oxygen using an Airstone. Move the slices to the beaker containing ACSF with the protease solution to perform mild digestion at room temperature. Following the digestion, wash out the protease by moving slices back to the beaker containing ACSF with activity blocker solution for five to 10 minutes at room temperature.Keep bubbling oxygen. Next, prepare a 100-millimeter Petri dish containing ACSF with 1%FBS at room temperature and move individual sections into the disk for microdissection. Under a fluorescent dissections scope, use a pair of fine forceps to micro dissect areas and layers of interest having a minimum of 10 to 50 cells.Using a Pasteur pipette, move the micro-dissected pieces to a 2-milliliter microcentrifuge tube containing approximately 0.8 milliliters of 1%FBS in ACSF solution. Titrate the dissected tissue in the micro-fuge tube at room temperature. Perform approximately 10 strokes with each of the flamed Pasteur pipettes starting with the biggest and ending with the smallest exit diameter.Dispense the dissociated cells into a 100-millimeter Petri dish containing oxygenated ACSF and wait five to seven minutes for the cells to gradually settle. Observe the GFP and RFP signal under a dissection microscope. Identify debris by examining the morphology under bright-field illumination.Once an area of cells with little debris has been identified, use the capillary and attach to tubing to pick cells by blocking the end of the tubing valve with the tongue. Then position the capillary close to the cells. Release the block on the capillary and quickly block again to capture the cell using capillary action.Move the capillary to a 100-millimeter Petri dish with fresh oxygenated ACSF and gently blow into the tube while observing the capillary tip under fluorescence optics to dispense the cells into the dish. Repeat the collection procedure until 100 to 150 cells are collected making sure that contaminants, such as debris, are minimal in bright-field differential interference contrast optics. Finally, using a fresh micro-capillary pipette each time, choose a single cell and expel it in no more than 0.5 microliters into a 0.2 microliter micro-fuge tube containing one microliter of sample-collection buffer with primers.Break the pipette tip in the micro-fuge tube to ensure that the cell stays in the collection buffer. Freeze the cells by putting the tubes on dry ice. Store in a minus 80-degree Celsius freezer until processing for RNA amplification.GABAergic neurons were isolated from the cingulet cortex of the mouse brain. RNA amplified from the manually sorted neurons ranged between 200 to 4, 000 base pairs in size with a peak distribution slightly above 500 base pairs. After bead purification, the cDNA library is further size restricted by a second round of purification using beads to eliminate fragments less than 200 base pairs and for the peak at approximately 350 base pairs.Sequencing showed a 4.8 times 10 to the 5th median or 6.9 times 10 to the 5th averaged mapped reads per cell. After duplicate RNA removal using UMIs, each single cell had a 1.0 times 10 to the 5th median or 1.4 times 10 to the 5th average unique reads per cell. In each single cell external RNA controls consortium, spike in RNA was used as an internal control to calculate absolute number of molecules added to the sample.There was a linear relationship of input to observed counts with a slope of 0.92 and adjusted R square of 0.94. An average of around 10, 000 genes per single neuron were detected using this procedure with more than 95%of the single cells detecting more than 6, 000 genes. After its development, this technique paved the way for molecular biologists to explore the transcriptome of distinct well-characterized mouse cortical interneurons, and identified select gene categories that are responsible for encoding the cellular identity.

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