The overall goal of this experiment is to generate barcoded genomic sequencing data from tens of thousands of single cells using a series of microfluidic devices. This method achieves unparalleled single-cell sequencing throughput. We use microfluidics to encapsulate, lyse, and barcode cells individually, preserving underlying genomic heterogeneity, which is lost in conventional shock in sequencing studies.
The main advantage of this technique is that it is high throughput and capable of producing single-cell data from tens of thousands of cells. Although we work with microbes in this demonstration, the workflow can be adapted for use with different cell types through minor modifications to the microfluidic device dimensions. To begin the protocol, prepare one milliliter of 3%weight per volume low-melting temperature agarose in 1X Tris-EDTA, or TE, buffer.
Keep the agarose solution on a 90 degree Celsius heat block just until syringe loading. Resuspend cells in one milliliter of phosphate-buffered saline, or PBS, and spin the cells. Aspirate the supernatant, and resuspend the cell pellet in one milliliter of 17%volume to volume density gradient medium in PBS, and keep the cells on ice until the syringe loading.
Next, load a three-milliliter syringe with fluorinated oil, or HFE, containing a 2%weight per weight PFPE-PEG surfactant. Fit it with a 27-gauge needle, and place it into a syringe pump. Load the cell suspension and molten agarose into one-milliliter syringes, both fit with 27-gauge needles, and place them into syringe pumps.
Then, set a space heater to high, and position the heating surface 10 centimeters away from the syringe. Ensure that the temperature measured at the syringe is approximately 80 degrees Celsius. Before inserting the tubes into the device, prime the pumps to remove the air from the line.
Connect the syringe needles to the microfluidic device inlets using pieces of polyethylene, or PE, tubing. Connect a piece of tubing to the outlet, and place the free end in a 15-milliliter collection tube. After dropmaking, place the collection tube at four degrees Celsius for 30 minutes to ensure the complete gelation of the agarose.
Remove the lower layer of oil from the collection tube using a three-milliliter syringe fitted with a 20-gauge needle, taking care not to disturb the top layer of the agarose droplets. Add one milliliter of 10%volume per volume PFO in HFE to break the agarose drops from their surfactant layer. Pipette the solution up and down for one minute to thoroughly coat the emulsions, which should be homogeneous and free of clumps.
Spin the conical tube at 2, 000 times g for one minute to collect the agarose microgels. Aspirate the PFO/HFE supernatant, and ensure that the microgels are now free of their surfactant layer and are clear. After washing the microgels, verify the cell encapsulation in the microgels under a microscope at a 400X magnification by staining a 10-microliter aliquot of gels with 1X nucleic acid stain.
Plug the cells inlet of a co-flow dropmaker device with a small piece of lead solder. Before inserting the tubes into the device, prime the pumps to remove the air from the line. Connect a piece of tubing to the outlet, and place the free end in a 0.2-milliliter PCR tube.
Use the flow rates on the screen for dropmaking. Collect the drops into the PCR tubes with approximately 50 microliters of drops in each tube. After the dropmaking, carefully remove the lower layer of HFE oil from the emulsions using gel-loading pipette tips, and replace it with FC-40 fluorinated oil containing a 5%weight per weight PFPE-PEG surfactant.
Then, program the thermal cycle. Before inserting the tubes into the device, prime the pumps to remove the air from the line. Connect the syringes containing HFE, tagmentation mix, and microgels to the microfluidic device inlets using pieces of PE tubing.
Connect a piece of tubing to the outlet, and place the free end in an empty one-milliliter syringe with the plunger drawn to the one milliliter line. Next, verify the microgel encapsulation rate under a light microscope at 400X magnification. Fit the syringe containing the tagmentation emulsions with a needle, and incubate it upright in a heat block or oven for one hour at 55 degrees Celsius to fragment the genomic DNA.
Prepare the barcode droplets for the merger by replacing the FC-40 oil fraction with HFE 2%weight per weight PFPE-PEG. Carefully transfer the drops into a one-milliliter syringe, fit it with a needle, and place it into a syringe pump. Load the incubated and tagmented microgel droplet syringe into a syringe pump.
Next, connect the three sodium chloride syringes to the two electrode inlets and single moat inlet using pieces of PE tubing. Before inserting the tubes into the device, prime the pumps to remove the air from the line. Connect the three HFE syringes mounted on pumps to the two spacer oil inlets and dropmaking oil inlet using pieces of PE tubing.
Then, shoot all droplet reinjection tubing with an antistatic gun before connecting the tubing to the syringe needles. Connect the PCR mix syringe, microgel drop syringe, and barcode drop syringe to their respective inlets with PE tubing. Connect the needle of the electrode syringe to a cold cathode fluorescent inverter using an alligator clip.
Set the inverter's DC power supply to two volts. Run the double merger device with the recommended flow rates. Collect the drops into PCR tubes with approximately 50 microliters of emulsion in each tube.
Prior to the thermal cycling, carefully remove the lower layer of HFE oil from the emulsions using gel-loading pipette tips, and replace them with FC-40 fluorinated oil containing a 5%weight per weight PFPE-PEG surfactant. Then, program the cycle. To recover the DNA from the thermal cycle droplets, pool the droplets into a microcentrifuge tube, and break the emulsions using 20 microliters of PFO.
Vortex them for 10 seconds to mix. Spin the tube. Carefully remove the upper aqueous layer from the tube using a pipette, and transfer it to a new microcentrifuge tube.
Discard the oil phase. Then, finally proceed to the library preparation, sequencing, and analysis steps. A histogram of the barcode counts versus the group size shows that a significant portion of the valid barcode groups is just above the 7.5 kilobase pairs per group threshold size, which excludes the PCR-mutated orphans.
The relative abundance of the cell types from a synthetic 10-cell community of three gram-negative bacteria, five gram-positive bacteria, and two yeasts was calculated by counting the raw reads and the barcode groups. A circular coverage map of B.subtilis reads from all barcode groups illustrates the uniformity of SiC-seq reads, with no observable dropout regions, where the average coverage was 5.55X. The uniformity of coverage was verified with a frequency distribution of normalized coverage values.
While attempting this procedure, it is important to maintain an encapsulation rate of around one barcode or cell for every 10 droplets. This limits the likelihood of a double encapsulation event, which can convolute the single cell data. After watching this video, you should have a good understanding of how to set up and operate the microfluidic devices used to generate single-cell sequencing data.
Once mastered, this technique can be run in eight hours or two four-hour days.
