Transcriptome Analysis of Single Cells

1. Cell Culture

Our lab uses primary neuronal rat hippocampal cultures for the experiments below. The following describes the modified Banker protocol for how these cell cultures are created and maintained.⁹ There are, of course, an exhaustive number of permutations of these cell culturing techniques and any established method tailored to the specific needs of a particular laboratory that provides a consistently healthy supply of cells would be a suitable substitute.

1. Five autoclaved, round, 12 mm coverslips are placed in a 35 mm dish and coated with 80 μg/mL Poly-D-Lysine (MW 70-150,000) in borate buffer O/N, rinsed with water (cell culture grade), then coated with 1 μg/mL Laminin in borate buffer O/N then rinsed again with water. NG media (MEM with Earle’s salts and GlutaMAX supplemented with glucose (0.6% w/v), penicillin (100 U/mL), streptomycin (100 μg/mL), horse serum (10% v/v)) is added and dishes are stored in an incubator (5% CO₂ at 37°C). The PDL150/laminin serves as a substrate for the isolated neurons to grow on as a monolayer. The coverslips can be coated up to two weeks before plating.
2. On the day of plating, primary neurons from embryonic day 18 rat hippocampi are plated in NG media by adding 1.5 mL of a 100,000 cells/mL suspension. Four to six hours after plating, each 35 mm dish is treated with 0.75 μL of 5 mM cytosine beta-D-arabinofuranoside (ara-C) to prevent growth of any contaminating glial cells. Twenty-four hours later, the plating media is changed to MEM with Earle’s salts and L-glutamine supplemented with 0.6% w/v glucose, 1 mM sodium pyruvate, and B27. Cells are permitted to grow for at least two weeks before use in any experiments to allow complete development of processes.

2. Preparing Pipettes

Note on RNases: The skin, saliva, and even breath are major sources of RNases, enzymes that degrade RNA. It is imperative that RNase-free technique be observed from this point on in the protocol so that the sample does not become contaminated with RNases and subsequently degrade. This includes always wearing gloves when handling the samples and reagents, never talking over samples or reagents, and using new boxes of sterilized pipette tips and tubes. Often, pipettes that are not designated exclusively for RNA work are decontaminated by wiping them down with an RNase treatment solution such as RNase AWAY (Molecular Bioproducts). However, these solutions will inhibit any downstream enzymatic reactions so it is also important to prevent contamination of your samples with these treatments.

1. Autoclave 1.5 mm outer diameter (O.D.) glass pipettes.
2. Using a micropipette puller, pull pipettes to a diameter appropriate for electrophysiological recordings, which may vary based on your puller, filament, and pipettes.¹⁰ The bore size is not too critical since the tip will be broken prior to cell collection.
3. Carefully store the pipettes in a dust-free environment where the tips will remain intact (ideally use a micropipette storage jar).
4. To break the tip in a controlled fashion, hold a kimwipe taut between your fingers in one hand and very gently brush the tip of the pipette across the paper 1 to 2 times. The opening should be approximately 75-100% of the target cell size. Adjust this step until you achieve the desired result.

3. Preparing Culture, Tubes, and Microscope

1. Prepare desired number of 1.7 mL Eppendorf tubes. Add 2 μL of PBS, first strand buffer if material is to be used for single cell amplifications, or any other solution for storage of the harvested material.
2. For harvesting, you will need a light microscope with a 40x objective fitted with a micromanipulator. Use a pipette holder with flexible tubing leading away from the holder. Affix the tubing to a stable surface to prevent unwanted movement of the pipette during collection. At the end of the tubing, insert a needle so that a 1 mL syringe with a Luer-Lock connection can be attached and changed in between users.
3. If using coverslips, work with one coverslip at a time. If necessary, transfer a single coverslip to a new 35 mm dish* containing PBS or media of choice. The longer the cells are out of the incubator, the more unhealthy they will become and the more their mRNA profiles will deviate from that of a healthy cell. It is critical to preserve the health of the cells on the other coverslips by keeping them in the incubator when not working with them. Work as quickly as possible with cells outside of the incubator.

*With our setup it is necessary to use the lid of a 35 mm dish since the walls of the actual dish are too high to allow proper advancement of the micropipette towards the coverslip.

4. Harvesting Cells

1. Secure the micropipette in the micropipette holder. Affix micropipette holder to the insert on the micromanipulators. Set the 40x objective.
2. Place the dish on the microscope stage and find a region of cells suitable for harvest. In the case of primary neuronal cultures, the cell should be relatively isolated from its neighbors to prevent harvest of surrounding cells and/or processes. The population of mRNA transcripts between the soma and processes vary, so if interested in somatic mRNAs solely, care should be taken to prevent contamination of the soma with processes. Place the cell you wish to harvest in the center of the field of view.
3. Use the micromanipulator to advance the micropipette toward the solution in the dish into the light path. Lower the pipette tip into the solution. Apply positive pressure from this point on by blowing lightly through the syringe. Look through the eyepiece. The pipette should appear as a shadow in the field of view. Well above the plane of the cells, adjust the position of the pipette tip until it is within the field of view and focus on the pipette tip using the coarse focusing knobs. At this point, it should be assessed if the bore size of the pipette is too large or too small. Practice harvesting will provide the ability to determine if the correct bore size has been achieved at this point.
4. Now, advance the pipette toward the plane of the cells. It is important that the tip is not advanced too hurriedly, causing it to break in the region from which you wish to collect. To prevent this, use the coarse focus to advance the focal plane BEFORE advancing the pipette tip towards the cells using the micromanipulators. When you are close to the cells, use the fine focus until both the cells and the pipette tip are in focus. Stop applying positive pressure before you reach the plane of the cells to prevent blowing them off of the coverslip.
5. Use the micromanipulators to position the pipette tip so that it is touching the cell soma you wish to harvest.
6. Using the syringe, apply gentle suction by mouth until the cell enters the pipette tip. If the cell is not entering the pipette, gently move the tip closer to the cell and downward until it lifts from the coverslip.

5. Saving the Cell

1. Use the micromanipulator to immediately move the pipette up and out of the solution.
2. Remove the pipette from the holder.
3. Hold the 1.7 mL Eppendorf tube in one hand and gently break the tip of the pipette on the side of the tube toward the bottom. (Aim for the 0.1 mL mark.)
4. With the broken tip centered inside the tube, insert a needle attached to a 1-3 mL syringe into the top opening of the pipette. Rapidly depress the plunger forcing the solution in the pipette to spray out into the tube. The cell should remain in the very tip of the pipette and this should be adequate to properly expel the cell. Be careful not to touch the tip to any liquid on the sides of the tube as capillary action will bring the liquid back into the pipette.
5. Quickly spin down the contents of the tube using a desktop microfuge and immediately freeze (store at -80°C) or place on ice for further processing (preferable).

6. aRNA Amplification

1. Round 1 First strand cDNA synthesis:
   Create mRNA/cDNA hybrids.
   1. For every 5 μL of single cell collection volume, add 1x of the following to the collection tube (on ice). The reaction can be scaled up for larger collection volumes (2x for 10 μL collection volumes, etc.). Create a master mix by multiplying the following reagents by the number of collection tubes plus 10-20% to account for pipetting error. Do this for every subsequent reaction in the aRNA ampification procedure.
      • ON ICE
      • 1.2 μL dNTP’s (2.5 mM each)
      • 2.4 μL 5x First strand buffer
      • 0.3 μL T7-oligo(dT) primer (100 ng/μL)
      • 1.2 μL DTT (100 mM)
   2. Bring the volume up to 10.25 μL with nuclease free water. Pipette mix and spin briefly using a microfuge.
   3. Incubate for 5 minutes at 70°C to denature any secondary structure of the mRNA. Immediately place on ice for at least 5 minutes.
   4. Add (again using a master mix):
      • 0.3 μL RNasin (40 U/μL)
      • 0.45 μL Superscript III (200 U/μL)
      • 1 μL nuclease free water
   5. Pipette mix and spin briefly. Incubate for 1 hour at 42°C.
   6. Incubate at 70°C for 15 minutes to inactivate the Superscript. Continue on to the next step.
2. Round 1 Second strand cDNA synthesis
   Create RNA primers from the mRNA portion of the mRNA/DNA hybrid to aid in synthesis of double stranded cDNA.
   1. • ON ICE
      • To the 12 μL first strand reaction, add:
      • 8 μL nuclease free water
      • 5.6 μL 5x Second strand buffer
      • 0.75 μL dNTP mix (2.5 mM each)
      • 0.25 μL DNA ligase (10 U/μL)
      • 1 μL DNA polymerase I (10 U/μL)
      • 0.25 μL Rnase H (2 U/μL)
      Mix thoroughly by pipetting and spin briefly. Incubate for 2 hours at 16°C.
   2. Add: 1 μL T4 DNA polymerase (5 U/μL). Mix by pipetting and spin briefly. Incubate 10 more minutes at 16°C.
   3. Clean up the reaction using the Qiagen MinElute kit as per the manufacturer’s instructions with slight modifications: ¹¹ Wash 2 times with 500 μL wash buffer instead of 1 time with 750 μL. Elute in nuclease free water.
   4. Concentrate to 2-4 μL using a Speedvac or by ethanol precipitation. For the ethanol precipitation, the glycogen acts as a nucleic acid carrier and is used when precipitating small amounts of DNA or RNA. The sodium from the sodium acetate helps neutralize the negative charge of the DNA backbone, aiding in precipitation. It is not necessary to make a master mix for routine precipitations.
      • Add 40 μL DEPC water
      • 1 μL glycogen (3 mg/mL)
      • 1/10 volume (3 μL) 3M Sodium acetate
      • 2.5 volumes (~125 μL) cold 100% ethanol
      Mix well. Precipitate at -80°C (30 min. to O/N).
   5. Centrifuge for 20 minutes at 4°C.
   6. Remove the supernatant and wash the pellet with 800 μL 70% ethanol. Be sure to fully dislodge the pellet either by pipetting or vortexing to aid in removal of excess salt.
   7. Centrifuge for another 20 minutes at 4°C. Remove the supernatant and air.
   8. Resuspend in 4 μL of nuclease free water. Store at -20 or -80 °C or continue on to the next step.
3. Round 1 In vitro transcription (IVT):
   Synthesize antisense RNA from the T7 promoter incorporated into the double stranded cDNA.
   1. This reaction is performed using the Ambion MEGAscript T7 kit as per the manufacturer’s instructions except scaled for a 10 μL instead of a 20 μL reaction.¹² It is imperative to assemble this reaction at room temperature and to keep the buffer at room temperature during assembly. The supplied buffer will precipitate the DNA if it is ice cold. Keep the NTPs and enzyme mix on ice when not in use.
      • AT ROOM TEMPERATURE
      • Transfer the resuspended double stranded cDNA to a thin walled PCR tube.
      • Add 4 μL NTP mix (18.75 mM each)
      • 1 μL 10x reaction buffer
      • 1 μL 10x enzyme mix
   2. Incubate 14 hours at 37°C in a thermocycler (preferable) or incubator.
   3. This reaction can be cleaned up using two different methods followed by concentration of the sample using a Speedvac or ethanol precipitation. For cleanup using a kit, proceed to Method A. For clean up with a standard phenol/chloroform extraction, proceed to Method B.
   4. Method A:
      1. Clean up the reaction using the AMBION Megaclear kit as per the manufacturer’s instructions with some modifications¹³: Wash 2 times with 500 μL wash buffer instead of 1 time with 750 μL. For the elution step, add 50 μL of nuclease free water to the center of the column, incubate at 70 °C for 10 minutes. Spin at 10,000g for 1 min. Repeat in a fresh tube. Combine eluates. Concentrate sample to 2-4 μL using a Speedvac OR by ethanol precipitation.
      2. For ethanol precipitation: Ammonium acetate is used in place of sodium acetate in this case because it is efficient at preventing co-precipitation of free nucleotides with the nucleic acid. This is important because of the high concentration of NTP’s used in the IVT reaction, which can inhibit downstream reactions.
         • 2 μL glycogen (5 mg/mL)
         • 0.1 volumes (10 μL) 5M Ammonium acetate
         • 2.5 volumes (~275 μL) cold ethanol
      3. Precipitate at -80°C (30 min. to O/N).*
      4. Centrifuge for 20 minutes at 4°C.
      5. Remove the supernatant and wash the pellet with 800 μL 70% ethanol (in nuclease free water). Be sure to fully dislodge the pellet to remove all of the excess salt.
      6. Centrifuge for another 20 minutes at 4°C.
      7. Remove supernatant and air dry.
      8. Resuspend pellet in 4 μL nuclease free water.
   5. Method B:
      1. Alternatively, the aRNA can be cleaned up with a standard phenol/chloroform extraction.
         • Add 90 μL DEPC water
         • 2 μL glycogen (5 mg/mL)
         • 0.1 volumes (10 μL) 5M Ammonium acetate
         • 1 volume (100 μL) phenol:chloroform:isoamyl alcohol 25:24:1 (equilibrated to pH 7.8-8.0)
      2. Vortex for 15 seconds. Centrifuge for 1.5 minutes at half maximal speed in a table top microcentrifuge. Transfer the top aqueous layer to a new tube containing 2.5 volumes (245 μL) of cold ethanol.
      3. Precipitate at -80°C (30 min. to O/N).
      4. Centrifuge for 20 minutes at 4°C.
      5. Remove supernatant and wash pellet with 800 μL 70% ethanol (in nuclease free water). Be sure to fully dislodge the pellet to remove all of the excess salt.
      6. Centrifuge for another 20 minutes at 4°C.
      7. Remove supernatant and air dry.
      8. Resuspend pellet in 4 μL nuclease free water.
         
         *The sample may freeze at this temperature during overnight incubations. It has been shown that this can actually increase yield of nucleic acids.

4. Round 2 First strand cDNA synthesis
   1. • ON ICE
      • Add 1 μL Random primers (0.05 mg/mL)*
   2. Heat at 70°C for 10 minutes. Immediately place on ice for at least 5 minutes.
   3. • Add 2 μL 5x First strand buffer
      • 1 μL DTT (100 mM)
      • 0.5 μL dNTP’s (2.5 mM each)
      • 0.5 μL RNasin (40 U/μL)
      • 1 μL Superscript III (200 U/μL)
   4. Mix thoroughly by pipetting and spin briefly. Allow to sit at room temperature for 10 minutes. This step is required to allow extension of the short random primers before the reverse transcription reaction is performed.
   5. Incubate for 30 minutes at 42°C.
   6. Heat 5 minutes at 95°C to denature the RNA in DNA/RNA hybrids. Place on ice for at least 5 minutes. Store at -20°C or -80°C or immediately continue on to the next step.
      
      *The concentration of random primers is important. If the concentration is too high, the product will be more truncated with each round of amplification.
5. Round 2 Second strand cDNA synthesis
   1. • ON ICE
      • Add 2 μL T7-oligo(dT) primer (10 ng/μL)*
   2. Heat for 5 minutes at 70°C. Immediately place on ice for at least 5 minutes. Spin briefly.
   3. • Add 43.5 μL nuclease free water
      • 15 μL 5x Second strand buffer
      • 1.5 μL dNTP mix (2.5 mM each)
      • 2 μL DNA polymerase I (10 U/μL)
   4. Mix thoroughly by pipetting and spin briefly. Incubate for 2 hours at 16°C.
   5. Add 2 μL T4 DNA polymerase (5 U/μL)
   6. Mix thoroughly by pipetting and spin briefly. Incubate 10 more minutes at 16°C.
   7. Clean up the reaction using the Qiagen Minelute kit as in Section 6.2.3.
   8. Concentrate to 2-4 μL with a Speedvac or ethanol precipitation as in Sections 6.2.4 to 6.2.8.
      
      *Note the different stock concentration of T7-oligo(dT) primer compared to first round second strand cDNA synthesis.
6. Round 2 In vitro transcription
   Perform as in Section 6.3.
   1. Repeat Sections 6.4 to 6.6 desired number of times. Note that each subsequent round of amplification results in shorter aRNA products. The number of rounds of amplification should be limited for this reason. Normally, two to three rounds of amplification are sufficient to amplify material harvested from a single cell to perform microarray analysis. In the case of microarray analysis, the third round IVT reaction is performed using the Illumina TotalPrep RNA Amplification Kit as per the manufacturer’s instructions. This procedure incorporates biotin-labeled UTP into the aRNA that is then used for detection on a microarray chip.
   2. Measure the concentration of third round aRNA using a Nanodrop or Agilent Bioanalyzer with a Nano RNA kit.

7. Representative Results

Successful harvest of a single cell from primary neuronal cultures can be completed in less than 2 minutes, depending on aptitude (see Figure 1). However, time for harvest will vary between systems and with intervening experimental manipulation. Subjecting the single cell to the aRNA procedure (see Figures 4A and 4B) results in microgram amounts of total amplified aRNA and produces a characteristic broad peak when analyzed with a bioanalyzer (see Figure 3). Three rounds can be completed in a minimum of three days, allowing for quick analysis of single cell gene expression.

Figure 1. Shown is an example of a successful harvest of an isolated neuron. We selected a relatively low-density region (A) and advanced the pipette tip toward the desired cell (B). The third image (C) shows the field of view after the cell had been harvested. Note that the surrounding processes remain on the coverslip.

Figure 2. Shown are two images of pipette tips, which are an inappropriate size for effective harvesting. These tips will lead to incomplete harvest (A) and harvest of surrounding mileu (B) respectively.

Figure 3. Following harvest and amplification, analysis using a bioanalyzer is recommended to examine the distribution and quantity of amplified RNA. A successful amplification from single cell material will yield total amounts in the low micrograms and will have a distribution that is smooth and wide.

Figures 4. Schematics of the first round (A) and the second round (B) of the aRNA procedure are shown.