Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

All animal experiments were approved of by the Pasteur Institute Safety Committee in accordance with the French Agriculture Ministry and the EU guidelines.

1. Prepare a 96-well Single-cell Sorting Plate

1. Prepare pre-amplification mix in a 1.5 ml tube by adding 5.0 µl of specific retro-transcription buffer, 1.3 µl of low ethylenediaminetetraacetic acid (EDTA) TE buffer (10 mM TE solution, pH 8, and 0.1 mM EDTA solution; 0.2 µM filtered), and 0.2 µl of Taq DNA polymerase per well (Figure 1a).
2. On a 96-well plate, distribute 6.5 µl of pre-amplification mix in each well (up to 48 wells). This plate is the 96-well single-cell sorting plate.
   NOTE: Using an electronic pipette is recommended for this protocol. It allows for precise and reproducible volume measurements and saves time.

Figure 1: 96-well plate loading procedure. The loading of the 96-well plates should be done very carefully, as it will impact the rest of the experiment. (a) On the 96-well single-cell sorting plate, the pre-amplification mix is distributed first, followed by the 0.2x assay mix. (b) The record of each single-cell position should be kept on a spreadsheet. A well without a cell is called a "no input" well and can be used as a control. Two rows can be spared to control primer efficiency with cDNA dilution (sequential one-in-ten dilutions from the equivalent of 10⁵ cells to one cell). (c) On the 96-well assay plate, the assay loading reagent is distributed first, followed by the addition of the primers. Do not forget to keep a layout of each primer position. Please click here to view a larger version of this figure.

3. Prepare a 0.2x assay mix in a 1.5 ml tube by adding 1.4 µl of each primer (primers 20x; up to 48 different probes; see Table 1). Adjust the final volume to 140 µl with low EDTA TE buffer.
4. Distribute 2.5 µl of the 0.2x assay mix to the 48 wells in the 96-well single-cell sorting plate containing the pre-amplification mix.
   NOTE: The final volume in each well of the 96-well sample plate should be 9 µl.
5. Seal the plate with a cover film. Vortex the plate and spin it down at 280 x g for 45 sec. Store the 96-well single-cell sorting plate at -20 °C or use it immediately.

2. Single Cell Dissociation

1. Using a CO₂ delivery system, euthanize a mouse by hypoxia. Fix the mouse on a dissection plate and open the peritoneal cavity longitudinally using scissors.
2. Recover ILCs from the liver.
   1. Prior to liver isolation, flush the liver-circulating cells.
      1. To bring out the portal vein, move the small and the large intestine to the left side of the mouse; the portal vein appears as the largest vein in the peritoneal cavity.
      2. With a 10 ml syringe and a 0.45 mm needle, flush the liver with phosphate-buffered saline medium (PBS) through the portal vein. To facilitate liver flushing, cut the gastric artery with scissors.
      3. To remove the gallbladder, pinch the base of the gallbladder with forceps and separate it from the liver with scissors.
      4. To isolate the liver, pinch the base of the liver with forceps, and using scissors, separate the liver from the rest of the peritoneal cavity.
      5. Transfer the liver in a potter tube with 5 ml of Roswell Park Memorial Institute medium (RPMI) 2% fetal calf serum (FCS).
3. Dissociate the liver with a mechanic dissociation using a pestle. Transfer the medium to a 15 ml tube. Bring the total volume to 14 ml with RPMI 2% FCS. Leave the cell suspension on ice for 15-20 min to decant the hepatocytes.
4. Recover the supernatant (without the hepatocytes) in a new 15 ml tube using a 1 ml pipette (12 ml of supernatant can be expected). Spin down at 120 x g for 7 min at 10 °C. Discard the supernatant after centrifugation.
5. Resuspend the pellet obtained in step 2.4 in 14 ml of density gradient medium (e.g., Percoll 40%). Spin down at 600 x g for 20 min at 20 °C. Remove the supernatant by aspiration.
6. Resuspend the pellet in 1 ml of potassium acetate (ACK). Leave at room temperature for 1 min. After 1 min, bring the total volume to 14 ml with Hank's Balanced Salted Solution (HBSS) 2% FCS. Spin down at 120 x g for 7 min at 10 °C. Discard the supernatant.
7. Stain the cells.
   1. Resuspend the pellet in 300 µl of biotinylated antibody mix (see the Materials Table; add all biotinylated antibodies mentioned) and transfer the cells in to 1.5 ml tube. Leave it in the dark for 20 min at 4 °C.
   2. Bring the total volume to 1 ml with HBSS 2% FCS. Spin down at 120 x g for 7 min at 10 °C. Resuspend the pellet in 300 µl of fluorochrome-coupled antibody mix and fluorochrome-conjugated streptavidin (see the Materials Table; add all fluorochrome-coupled antibodies mentioned). Leave it in the dark for 20 min at 4 °C.
8. Bring the total volume to 1 ml with HBSS 2% FCS. Spin down at 120 x g for 7 min at 10 °C. Remove the supernatant using a 1 ml pipette. Resuspend the pellet in 1 ml HBSS and propidium iodide (Pi; 1:4,000).
   NOTE: When using the widely expressed ILC cell-surface markers, 3 populations are defined: NKp46⁺ IL-7Rα^–, NKp46⁺ IL-7Rα⁺, and NKp46^– IL-7Rα⁺. All populations are sorted lineage^– CD3^– CD4^– CD45.2⁺.

3. Single-cell Fluorescence-activated Cell Sorting (FACS)

1. Use FACS to sort single cells¹³.
   NOTE: Different cell types can be sorted on the same 96-well single-cell sorting plate (Figure 2).
   1. Use FACS to sort one cell per well containing the 0.2x assay mix and the pre-amplification mix on the 96-well single-cell sorting plate. Use a freshly prepared 96-well single-cell sorting plate or thaw it if stored at -20 °C.
      1. Put a sealed 96-well plate on the FACS plate holder. Use an empty 96-well plate as a test. Position the plate with the A1-well on the left and toward the experimenter.
      2. Adjust the plate holder to obtain a drop in the center of the A1-well with verification beads.
      3. Repeat step 3.1.1.2 with all the wells in line A.
      4. Remove the seal from the 96-well plate and sort 100 verification beads per well. Check for drop formation in the center of the wells.
      5. When properly adjusted, place the 96-well single-cell sorting plate on the plate holder. Draw the plate layout and sort 1 cell per well.
         NOTE: The proper positioning of each cell on the 96-well single-cell sorting plate is essential. A plate layout should be kept on a spreadsheet software. The plate layout will be used in the next several steps (Figure 1b)¹⁴. Leave one well containing 0.2x assay mix and pre-amplification mix on the 96-well sample plate without cells. This well is used as a no-input control. Leave two rows of 6 wells for a cDNA dilution. These wells are used as controls for primer efficiency (Figure 1b).
      6. Store the 96-well single-cell sorting plate at -20 °C or use it immediately.

4. Pre-amplification

1. Use a fresh or thawed 96-well single-cell sorting plate obtained in step 3.1.1.6. Vortex the plate and spin it down (280 x g for 45 sec).
2. Place the 96-well single-cell sorting plate on the thermocycler. Perform reverse transcription and pre-amplification as per the mentioned program in Figure 3 and Table 2.

Figure 3: Pre-amplification program. In order to have enough material, pre-amplification of specific target genes on sorted single cells is required. The 96-well single-cell sorting plate is loaded on a thermocycler to follow the pre-amplification program. The pre-amplification products are then diluted with low EDTA TE buffer and can be used immediately or frozen at -20 °C. Please click here to view a larger version of this figure.

3. Dilute the pre-amplified samples by adding 36 µl of low EDTA TE buffer into each well.
4. Seal the plate with a cover film. Vortex the plate and spin it down (280 g for 45 sec). Store the pre-amplified, 96-well single-cell sorting plate at -20 °C or use it immediately.

5. Prepare a 96-well Sample Plate

1. Prepare 191 µl of master mix by adding 175 µl of qPCR master mix and 17.5 µl of sample loading reagent.
2. On a new 96-well plate, distribute 3.6 µl of master mix in each well (up to 48 wells). This is the 96-well sample plate.
3. Transfer 2.9 µl of pre-amplified cDNA from the 96-well sample plate to the new 96-well plate. Keep the same position for each sample between the 96-single cell sorting plate and the 96-well sample plate.
4. Seal the plate with a cover film. Vortex the plate and spin it down (280 x g for 45 sec). Place the new 96-well plate on ice protected from light. Store the 96-well sample plate at -20 °C or use it immediately.

6. Prepare a 96-well Assay Plate

1. On a new 96-well plate, distribute 3 µl of assay loading reagent in each well (up to 48 wells). This plate is called the 96-well assay plate.
2. Add 3 µl of primers to each well. Keep a layout on a spreadsheet software (Figure 1c and Table 1; use all primers listed in Table 1). The proper positioning of each primer on the 96-well assay plate is essential for the next several steps.
   1. If the number of samples is below 48, add water instead of primers to unused wells.
3. Seal the plate with a cover film. Vortex the plate and spin it down (280 x g for 45 sec). Store the 96-well assay plate at -20 °C or use it immediately.
   NOTE: To avoid repeated freezing and thawing of the primers, distribute the primers for the pre-amplification mix and for the 96-well assay plate at the same time.

7. Single-cell Gene-expression Chip

1. Place the integrated microfluidic circuit (IFC) on the bench and check the valves using the capped syringe. Open the syringe, place it perpendicular to the valve, and press firmly. The O-ring should move. Fill the chip with control line fluid (0.3 ml of tuberculin).
2. Repeat step 7.1 with the second valve.
   NOTE: No liquid should be spilled over the chip.
3. Remove the blue film from the bottom of the chip. Load the chip into the IFC controller. On the IFC controller screen, select "PRIME" and then "RUN." The control of the microfluidic lines last 10 min.
4. Eject the chip and re-seal the blue film on the bottom of the chip.

Figure 4: Single-cell multiplex gene-expression chip loading. These steps require great precision, especially during the transfer of the 96-well plate to the single-cell multiplex gene-expression chip. To avoid loading errors and misplacements, it is highly recommended to work sequentially. The volume taken for each transfer should be controlled during the pipetting process. Finally, it is important to avoid any bubbles and to remove them in case of formation. Please click here to view a larger version of this figure.

5. Using an 8-channel pipette, transfer 5 µl from the 96-well assay plate on the A1 side (left side) of the chip. Fill the left side of the chip, as indicated in Figure 4. Be careful to always draw the same volume into the tips.
   1. Do not create bubbles. If bubbles appear, remove them using 10 µl tips. Change tips for each well of the chip.
6. Repeat step 7.5 on the right side of the chip using 5 µl from the 96-well sample plate.
7. Remove the blue film from the bottom of the chip. Load the chip into the IFC controller. On the IFC controller screen, select "RUN SCRIPT" and then "RUN." The loading of the microfluidic lines lasts 45 min.
8. Eject the chip and re-seal the blue film on the bottom of the chip.

8. Run the Chip

1. On the microfluidic qPCR computer, select the "Data Collection" software. Once it starts, select "New Run."
2. Select "Eject," remove the blue film from the bottom of the chip, and load the chip. Place the chip to get the A1 well on the left and toward the experimenter.
3. On "Project setting," select "None" and then "Next."
4. Select "New chip run," "New chip directory" (create a folder for the experiment), and "Next."
5. On "Gene expression" select, "Passive reference = ROX" (carboxy-x-rhodamine) and "Single probe;" select the correct filter according to the primers. Select "Next."
6. Select "Browse" and select the correct program according to the primers used.
   NOTE: "Default-10min-Hotstart.pcl" is the most commonly used program for single-cell multiplex RT-qPCR.
7. Select "Start Run". The reaction takes approximately 90 min.
8. Once finished, select "Eject-Done."

9. Data Analysis

1. Open the "Real-Time PCR Analysis" software, select "File" and "Open," find the experiment folder, and select "ChipRun.bml file."
2. Click on "Analysis View," "Results Table," and "Heat Map View;" boxes marked with an "X" are below the threshold detection level and/or had bad amplification curves.
3. Name the samples. Go to "Sample Setup" and select "New SBS 96." Click on "Mapping" and select "…" Copy and paste the sample layout design from the spreadsheet software. Define the pasted layout as "Sample Name."
4. Name the assays. Repeat step 9.3 with the primer names in "Detector Setup." Define the pasted layout as "Detector Name."
5. Click on "Analysis view" and "Analyze;" the sample and primer names will be attributed automatically to the samples and primers (Figure 7).
6. Calculate Ct, Delta Ct, and Fold Change for each sample. Use a housekeeping gene as an internal control (here, Gapdh):
   ΔCt = Ct_sample – Ct_{internal control}
   Fold Change = 2^{-(ΔCtsample-ΔCtnegative control)}
   1. Click on "Detector Setup" and select the well with no input control (use the housekeeping gene as an internal control to normalize gene expression). Select "Editor," "define reference," and "Update."
   2. Select all wells to which the above-defined internal control will be applied. Select "Editor" and "define as Test." Select an appropriate reference gene and click "Update."
   3. Select "Sample Setup" to select the negative control well. Select "Editor" and define "Reference." Click "Update."
   4. Select "Analysis View" and "Analyze;" the Delta Ct and Fold Change will automatically be calculated.
7. Export the data. Select "File" and "Export, save as "Table Results," select the destination folder, and hit "Save" and "Exit."
8. Repeat step 9.7, but instead of "Table Results," save as "HeatMap Results."