An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

1. Microfluidic Device Fabrication

1. Prepare the microfluidic mask designs using computer-aided design (CAD) software (provided as .DWG; see Supplementary Files). Have these designs printed by the vendor with a 10 µm resolution on a circuit board film.
   NOTE: For multi-layered microfluidic devices, the corresponding masks contain alignment marks.
2. For each device, fabricate the SU-8 master mold (Figure 1A) as follows.
   1. Prepare a 3-in diameter silicon wafer by pouring approximately 1 mL of SU-8 3025 photoresist onto the center of the wafer. Secure the wafer on the spin coater chuck by applying suction.
   2. See Table 1 for a listing of the layer thicknesses and spin speeds for each device. For all devices, begin the spin coating with 30 s at 500 rpm, followed by 30 s at the indicated speed.
   3. Remove the SU-8-silicon wafer from the spin coater and soft bake it on a hotplate set to 135 °C for 30 min. Allow the wafer to cool to room temperature after baking.
   4. Expose the SU-8-silicon wafer with the appropriate microfluidic mask under a collimated 190-mW, 365-nm UV LED for 3 min.
   5. After the exposure, hard bake the wafer on a hotplate set to 135 °C for 1 min. Following this baking step, allow the wafer to cool to room temperature.
   6. For a single-layered microfluidic device, skip to step 1.2.7. For a multi-layered microfluidic device, repeat steps 1.2.1 – 1.2.5 for the second layer of the photoresist (Figure 1B).
   7. Following the first hard bake for a single layer device (or the second hard bake for a multi-layered device), develop the wafer by immersing it into a bath of propylene glycol monomethyl ether acetate (PGMEA) for 30 min.
   8. After the wafer's development, use a squirt bottle containing PGMEA to rinse the wafer. Then rinse the wafer with a squirt bottle containing isopropanol before placing it on a 135 °C hotplate for 1 min to dry.
   9. Place the wafer (henceforth referred to as the master) into a Petri dish for casting with polydimethylsiloxane (PDMS).
3. With the master prepared in step 1.2, proceed to perform the device fabrication with a PDMS casting.
   1. Prepare the PDMS by combining a silicone base with a curing agent in an 11:1 ratio by mass. Mix the silicone base and curing agent by hand with a stir stick.
   2. Degas the PDMS by placing it into a degassing chamber and applying a vacuum. Allow the PDMS to degas until air bubbles are no longer visible (typically 30 min).
   3. Carefully pour the degassed PDMS over the master, to a final PDMS-layer thickness of approximately 5 mm. Degas the PDMS again to ensure the removal of any air bubbles.
   4. After the degassing, bake the PDMS and the master at 80 °C for 80 min.
   5. Carefully excise the cured PDMS slab from the baked master using a razor blade. Ensure that all cuts are on top of the silicon wafer.
      NOTE: Any cuts made off the silicon wafer may result in a lip preventing a uniform bonding.
   6. Punch the inlets and outlets using a 0.75 mm biopsy punch. Remove any dust and stray PDMS using a packaging tape on the feature side of the device.
   7. Prior to plasma treating the device, clean a 50 mm x 75 mm glass slide by rinsing it with isopropanol and drying it.
   8. For the plasma treatment, place the PDMS slab and glass slide into the plasma bonder with the features facing up. Perform the plasma treatment using 1 mbar O₂ plasma for 1 min. Bond the device to the glass slide by bringing the exposed, or face up, sides together.
   9. Following the plasma treatment, bake the device at 80 °C for 40 min.
   10. Finally, inject glass surface treatment fluid into one of the inlets to render the microfluidic channels hydrophobic. Ensure all channels are completely flooded with the solution and repeat the injection for each dropmaker. Bake the treated device at 80 °C for 10 min to evaporate excess solvent.

2. Encapsulation of Cells in Agarose Microgels

NOTE: See Figure 2A.

1. Prepare 1 mL of 3% w/v low-melting temperature agarose in 1x Tris-EDTA (TE) buffer. Keep the agarose solution on a 90 °C heat block until immediately prior to the syringe loading.
2. Prepare the cell suspension.
   NOTE: This protocol and its associated microfluidic devices have been validated to work with bacterial cells, either from a frozen stock or fresh preparation. Mammalian cells, depending on the cell type, may require an adjustment of the microfluidic channel dimensions to accommodate the larger cell sizes.
   1. Resuspend the cells in 1 mL of phosphate-buffered saline (PBS).
   2. Count the cells in a hemocytometer or by flow sorting. For 25 µm microgels with a target cell encapsulation rate of 1 in 10, prepare 1 mL of cell suspension at a final concentration of 2.4 x 10⁷ cells/mL.
   3. Spin the cells down at 3,000 x g for 3 min. Aspirate the supernatant and resuspend the cell pellet in 1 mL of 17% v/v density gradient medium (see Table of Materials) in PBS. Keep it on ice until the syringe loading.
3. Load a 3-mL syringe with fluorinated oil (HFE) containing a 2% w/w perfluoropolyether-polyethylene glycol (PFPE-PEG) surfactant, fit it with a 27 G needle, and place it into a syringe pump.
   NOTE: Fit all syringes with a 27 G needle for the microfluidic steps in this protocol. To reduce the risk of accidental pricking, keep caps on all needles until the pump operation begins.
4. Load the cell suspension and molten agarose into 1-mL syringes, fit both with 27-gauge needles, and place these into syringe pumps.
5. Keep the agarose syringe and pump warm with a small space heater to prevent the agarose from gelling in the syringe and inlet tubing. Set the space heater to HIGH and position it so that the heating surface is about 10 cm away from the syringe. Ensure that the temperature measured at the syringe is approximately 80 °C.
   NOTE: Users are advised to maintain the heater at the recommended distance from the pumping apparatus to reduce the risk of equipment damage, including melting of the tubing.
6. Generate 25 µm microgel drops using the co-flow dropmaking device.
   NOTE: See Figure 3A for a device schematic indicating the location of the reagent inlets and outlet.
   1. Connect the syringe needles to the microfluidic device inlets using pieces of polyethylene (PE) tubing. Before inserting the tubes into the device, prime the pumps to remove the air from the line.
   2. Connect a piece of tubing to the outlet and place the free end in a 15 mL collection tube.
   3. Use the following (recommended) flow rates for dropmaking: 800 µL/h for the HFE 2% w/w PFPE-PEG; 200 µL/h for the cell suspension in PBS; and 200 µL/h for the 3% w/v agarose.
7. After the dropmaking, place the collection tube at 4 °C for 30 min to ensure the complete gelation of the agarose.

3. Breaking and Washing the Agarose Microgels

1. Remove the lower layer of oil from the collection tube using a 3 mL syringe fitted with a 20 G needle, taking care not to disturb the top layer of the agarose droplets.
2. Break the emulsions with perfluorooctanol (PFO).
   1. Add 1 mL of 10% v/v PFO in HFE to the agarose drops. Pipet this solution up and down for 1 min to thoroughly coat the emulsions.
      NOTE: For all the microgel wash steps, pipet the solutions with a 1,000 µL tip. The microgel suspension should appear homogeneous and free of clumps after pipetting it.
   2. Spin the conical tube at 2,000 x g for 1 min to collect the agarose microgels. Remove the PFO/HFE supernatant by aspiration; the microgels are now free of their surfactant layer and will appear clear.
3. Wash the microgels with a surfactant in hexane.
   CAUTION: Hexane is a volatile organic solvent, and the wash in step 3.3 should be conducted in a fume hood.
   1. Add 2 mL of 1% v/v sorbitan monooleate nonionic surfactant (see Table of Materials) in hexane to the agarose microgels. Pipet up and down 10x to mix, ensuring the complete breakup of the microgel pellet.
   2. Spin the tube at 1,000 x g for 1 min to collect the microgels. Aspirate the supernatant to remove the surfactant/hexane solution.
   3. Repeat the surfactant/hexane wash.
4. Wash the microgels in an aqueous buffer to remove any residual organic solvent.
   1. Add 5 mL of TET buffer [0.1% v/v octylphenol ethoxylate nonionic detergent (see Table of Materials) in 1x TE] to the conical tube. Pipet up and down 10x to mix.
   2. Spin the conical tube at 2,000 x g for 2 min to collect the microgels. Aspirate the supernatant to remove the TET buffer.
   3. Repeat the TET wash 2x.
   4. Add 5 mL of 1x TE buffer to the conical tube. Pipet up and down 10x to mix.
   5. Spin the conical tube at 2,000 x g for 2 min to collect the microgels. Aspirate the supernatant to remove the TE buffer.
   6. Repeat the TE wash.
5. Verify the cell encapsulation in the microgels under a microscope at a 400X magnification by staining a 10 µL aliquot of gels with 1x nucleic acid stain (see Table of Materials).
   NOTE: Microgels will appear clear under the transparent channel while cell DNA will fluoresce under the GFP channel (497/520 nm excitation/emission wavelengths). An overlay of transparent and fluorescent channels will show the cells in the microgels as shown in Figure 4A.

4. Lysis of Cells in Agarose via Lytic Enzymes

1. Prepare 1 mL of a lytic enzyme cocktail using 800 µL of TE buffer (1x), 2 µL of yeast lytic enzyme (5 U/µL stock, 10 U/mL final), 30 µL of dithiothreitol (1 M stock, 30 mM final), 60 mg of lysozyme (lyophilized powder), 15 µL of EDTA (0.5 M stock, 7.5 mM final), 2 µL of mutanolysin (100 U/µL stock, 200 U/mL final), 2 µL of lysostaphin (10 U/µL stock, 20 U/mL final), and 30 µL of NaCl (1 M stock, 30 mM final).
2. Add additional TE to bring the volume to 1 mL. Mix the solution by vortexing.
3. Add the entire 1 mL of the lytic enzyme solution to no more than 1 mL of the washed microgels. Mix by pipetting 10x. Incubate the mixture at 37 °C for >2 h in a shaker (the maximum is overnight incubation).

5. Detergent-based Microgel Treatment

1. Following the lysis via lytic enzymes (step 4), wash the microgels.
   1. Spin down the microgels at 2,000 x g for 2 min and aspirate the supernatant. The droplets will appear opaque white due to cell debris dispersed in the pellet.
   2. Resuspend the microgels in 5 mL of 10 mM Tris-HCl buffer and pipet up and down 10x to mix.
   3. Spin the mixture down at 2,000 x g for 2 min, then remove the supernatant by aspiration.
2. Perform a detergent treatment on the microgels.
   1. Resuspend the gels in a lithium dodecyl sulfate (LiDS) lysis buffer (0.5% w/v LiDS in 20 mM Tris-HCl) and 60 µL of 0.5 M EDTA to a final volume of 3 mL.
   2. Add 5 µL of a Proteinase K enzyme (800 U/mL stock). Pipet up and down 10x to mix, then incubate the mixture at 42 °C on a heat block for 1 h to solubilize the cell membranes and digest the proteins.
3. Following the detergent treatment, wash the microgels.
   1. Spin the conical tube with the microgels down at 2,000 x g for 2 min. Remove the supernatant by aspiration.
   2. Wash the microgels with 10 mL of 2% v/v polysorbate 20 in water. Pipet up and down 10x to mix.
   3. Spin the conical tube down at 2,000 x g for 2 min, then remove the supernatant by aspiration.
   4. Wash the microgels with 10 mL of 100% ethanol to inactivate any residual enzyme. Pipet up and down 10x to mix.
   5. Spin the conical tube down at 2,000 x g for 2 min, then remove the supernatant by aspiration.
   6. Wash the microgels with 10 mL of 0.02% v/v polysorbate 20 in water. Pipet up and down 10x to mix.
   7. Spin the conical tube down at 2,000 x g for 2 min, then remove the supernatant by aspiration.
   8. Repeat the polysorbate 20 wash 3x. Pass the solution through a 100-µm cell strainer prior to the last wash to remove any large clumps.
4. Resuspend the microgels in 5 mL of 10 mM Tris-HCl buffer to prevent DNA degradation. The microgels may be stored at 4 °C for up to 1 week prior to the tagmentation (step 7).

6. Generating Barcode Droplets by Digital PCR

1. Prepare a 500-pM stock of BAR primer (Table 2) in a 1x TE buffer in a low-bind tube. Prior to each use, dilute the primer to a working stock of 1 pM and heat it to 70 °C for 1 min on a heat block.
2. Prepare a 150-µL PCR reaction mix using 75 µL of high-fidelity hot start master mix (see Table of Materials) (2x), 42 µL of PCR-grade water, 3 µL of DNA_BAR primer (10 µM stock, 0.2 µM final), 3 µL of P7_BAR primer (10 µM stock, 0.2 µM final), 6 µL of barcode dilution (1 pM stock, 40 fM final), 6 µL of polysorbate 20 (50% v/v stock, 2% final), and 15 µL of PEG 6k (50% w/v stock, 5% final). Mix by pipetting up and down 10x.
3. Prepare an HFE-backed syringe by drawing 200 µL of HFE oil into a syringe and fit it with a needle. Attach a section of PE tubing to the needle and prime the line by hand. Insert the end of the PE tubing into the target solution and carefully draw all 150 µL of the PCR mix into the PE tubing and syringe. Load the syringe into a syringe pump.
4. Load a 1 mL syringe with fluorinated oil (HFE) containing a 2% w/w perfluoropolyether-polyethylene glycol (PFPE-PEG) surfactant, fit it with a needle, and place it into a syringe pump.
5. Generate 25 µm barcode drops using the co-flow dropmaking device.
   NOTE: See Figure 3A for a device schematic indicating the location of the reagent inlets and outlet.
   1. Plug the Cells inlet of a co-flow dropmaker device with a small piece of lead solder.
   2. Connect the syringes loaded with HFE and PCR mix to the microfluidic device inlets using pieces of PE tubing, using the Molten Agarose inlet for the barcode PCR mix. Before inserting the tubes into the device, prime the pumps to remove the air from the line.
   3. Connect a piece of tubing to the outlet and place the free end in a 0.2 mL PCR tube. Use the following (recommended) flow rates for dropmaking: 600 µL/h for the HFE 2% w/w PFPE-PEG and 200 µL/h for the PCR mix. Collect the drops into the PCR tubes with approximately 50 µL of drops in each tube.
6. After the dropmaking, carefully remove the lower layer of HFE oil from the emulsions using gel-loading pipet tips and replace it with FC-40 fluorinated oil containing a 5% w/w PFPE-PEG surfactant. Thermal cycle with the following protocol: 98 °C for 3 min, 40x of (98 °C for 10 s, 62 °C for 20 s, 72 °C for 20 s), 72 °C for 5 min, and then hold at 12 °C.
   NOTE: The thermal-cycled droplets may be stored at 4 °C for up to 1 day.
7. Verify the barcode amplification and encapsulation rate.
   1. Prepare a 1x nucleic acid stain (see Table of Materials) in HFE with a 2% w/w PFPE-PEG surfactant; the stain is marginally miscible in HFE and will bind to the DNA in the droplets.
   2. Add 1 µL of thermal-cycled barcode emulsion to 10 µL of staining oil. Incubate them for 5 min at room temperature.
   3. Image droplets by fluorescent microscopy (GFP channel, 497/520 nm excitation/emission wavelengths) at a 200X magnification and count the barcode encapsulation rate. Note that the signal will be discrete: the droplets containing amplified barcodes will fluoresce brightly, whereas the empty drops will appear dark (Figure 4B).

7. Tagmentation of Genomic DNA in Droplets

NOTE: See Figure 2B.

1. Prepare 500 µL of tagmentation solution using reagents from a next-generation sequencing library preparation kit (see Table of Materials). Use 7 µL of tagmentation enzyme, 250 µL of tagmentation buffer, and 243 µL of PCR-grade water. Mix them by vortexing and spin the mixture down to collect. Load this solution into an HFE oil-backed 1 mL syringe and fit it with a needle.
2. Prepare the microgels for the reinjection.
   1. Spin down the microgel tube for 2 min at 2,000 x g and aspirate the supernatant. Transfer 200 µL of gels to the top of an HFE-backed syringe with a gel-loading pipet tip and seal the nozzle with a small piece of tape.
   2. Using a 3D-printed centrifuge adapter (see Supplemental Files 1 and 2), spin the microgel syringe for 3 min at 3,000 x g.
   3. Remove the liquid supernatant from the syringe using a gel-loading pipet tip. Push the microgel layer to the base of the syringe nozzle. Fit the syringe with a needle.
3. Load a 3 mL syringe with fluorinated oil (HFE) containing a 2% w/w perfluoropolyether-polyethylene glycol (PFPE-PEG) surfactant, fit it with a needle, and place it into a syringe pump.
4. Re-encapsulate the microgels into droplets containing tagmentation reagents.
   NOTE: See Figure 3B for a device schematic indicating the location of the reagent inlets and outlet.
   1. Connect the syringes containing HFE, tagmentation mix, and microgels to the microfluidic device inlets using pieces of PE tubing. Before inserting the tubes into the device, prime the pumps to remove the air from the line.
   2. Connect a piece of tubing to the outlet and place the free end in an empty 1-mL syringe with the plunger drawn to the 1 mL line.
   3. Use the following (recommended) flow rates for dropmaking: 2,000 µL/h for the HFE 2% w/w PFPE-PEG, 200 µL/h for the microgels, and 500 µL/h for the tagmentation mix.
5. Verify the microgel encapsulation rate under a light microscope at a 400X magnification. Approximately 80 – 90% of the drops should contain a microgel, as shown in Figure 4C.
6. Fit the syringe containing the tagmentation emulsions with a needle and incubate it upright in a heat block or oven for 1 h at 55 °C to fragment the genomic DNA.

8. Single-cell Barcoding by Microfluidic Double Merger

NOTE: See Figure 2C.

1. Prepare the barcode droplets for the merger by replacing the FC-40 oil fraction with HFE 2% w/w PFPE-PEG. Carefully transfer these drops into a 1 mL syringe, fit it with a needle, and place it into a syringe pump.
2. Load the incubated and tagmented microgel droplet syringe into a syringe pump.
3. Prepare 500 µL of PCR mix. Add the reagents in the following order to prevent precipitates from forming: 140 µL of PCR-grade water, 10 µL of P5_DNA primer (10 µM stock, 0.2 µM final), 10 µL of P7_BAR primer (10 µM stock, 0.2 µM final), 50 µL of PEG 6k (50% w/v stock, 5% final), 50 µL of polysorbate 20 (50% v/v stock, 5% final), 250 µL of Taq Master Mix (2x) (see Table of Materials), 10 µL of isothermal polymerase (see Table of Materials), and 10 µL of neutralization buffer (see Table of Materials). Mix them by pipetting up and down 10x and spin the mixture down to collect it. Load this solution into an HFE-backed 1-mL syringe, fit it with a needle, and place it into a syringe pump.
4. Load three 3-mL syringes with fluorinated oil (HFE) containing a 2% w/w perfluoropolyether-polyethylene glycol (PFPE-PEG) surfactant, fit each with a needle and place them into syringe pumps.
5. Load three 1 mL syringes with 2 M NaCl, fit each with a needle and set them aside.
6. Merge the barcode droplets, tagmented genome droplets, and PCR mix using the double merger device.
   NOTE: See Figure 5 for a device schematic indicating the location of the reagent and electrode inlets and outlet.
   1. Connect the 3 NaCl syringes to the 2 electrode inlets and single moat inletusing pieces of PE tubing. For the electrodes, apply pressure to the syringes manually until the electrodes are completely filled with salt solution. After filling the electrodes, apply manual pressure to the moat syringe until the moat is filled. Plug the free end of the moat with a small piece of lead solder.
   2. Connect the 3 HFE syringes mounted on pumps to the 2 spacer oil inlets and dropmaking oil inletusing pieces of PE tubing. Before inserting the tubes into the device, prime the pumps to remove the air from the line.
   3. Connect the PCR mix syringe, microgel drops syringe, and barcode drops syringe to their respective inlets using PE tubing. Shoot all droplet reinjection tubing with an antistatic gun before connecting them to the syringe needles; the antistatic treatment reduces the risk of droplet coalescence induced by static charges on the PE tubing.
   4. Connect a piece of PE tubing to the outlet and place the free end in a 0.2 mL PCR tube.
   5. 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 2 V.
   6. Run the double merger device with the recommended flow rates: 300 µL/h for the tagmented microgel drops, 100 µL/h for the barcode drops, 1500 µL/h for the HFE 2% w/w PFPE-PEG (dropmaking oil), 600 µL/h for the amplification mix, 200 µL/h for the HFE 2% w/w PFPE-PEG (barcode spacer oil), and 700 µL/h for the HFE 2% w/w PFPE-PEG (microgel spacer oil). Collect the drops into PCR tubes with approximately 50 µL of emulsion in each tube.
7. Prior to the thermal cycling, carefully remove the lower layer of HFE oil from the emulsions using gel-loading pipet tips and replace them with FC-40 fluorinated oil containing a 5% w/w PFPE-PEG surfactant. Thermal cycle with the following protocol: 65 °C for 5 min, 95 °C for 2 min, 30x (95 °C for 15 s, 60 °C for 1 min, 72 °C for 1 min), 72 °C for 5 min, then hold at 12 °C.
8. Recover the barcoded DNA from the thermal-cycled droplets.
   1. Pool the droplets into a microcentrifuge tube and break the emulsions using 20 µL of PFO. Vortex them for 10 s to mix.
   2. Spin the tube at 10,000 x g for 1 min to fractionate the mixture into aqueous (top) and oil (bottom) phases. Carefully remove the upper aqueous layer from the tube using a pipet and transfer it to a new microcentrifuge tube. Discard the oil phase.
   3. Purify the barcoded PCR product in a spin column according to the manufacturer's protocol and elute it in 20 µL of 1x TE buffer.
   4. Proceed to the performed sequencing and analysis as per steps 9 and 10.

9. Library Preparation and Sequencing

1. Prepare the single-cell library for the sequencing by following the manufacturer's protocols for fragment size-selection and quantification.
2. Sequence the paired-end library with default chemistry for Read 1 and Read 2. Use the custom Index 1 primer I7_READ (Table 2) for a 15-bp index read, corresponding to the single-cell barcode.

10. Single-cell Data Analysis

NOTE: Custom Python scripts for quality control and preliminary analysis of SiC-seq data can be downloaded from https://www.github.com/AbateLab/SiC-seq.

1. Run the script "barcodeCleanup.py" to perform quality control on the barcoded reads and export the single-cell data to an SQLite database. For a control experiment, use this script with the "-align" flag set to align the reads to known reference genomes.
2. Analyze the purity of the barcode groups (for a control experiment) using the script "purity.py" and confirm the high purity values consistent with Figure 6B.