TITLE: Quantification by Droplet Digital PCR (ddPCR)
AUTHORS: Shuchen Feng, Adélaïde Roguet

[STEPS]
?. When all reagents are thawed on ice, vortex Supermix, Reverse transcriptase and DTT throughly for 30 seconds. Vortex to mix primers and probes stocks.
?. Plate Set up: see sub-steps below.
This step requires to keep the mixture cold/on ice.
?. Droplet generation handling.
It is NOT required to keep the plate cold/on ice during this process.
?. Turn on the PX1 PCR plate sealer and let heat to 180ºC. Correctly place the plate support block, the ddPCR plate, foil cover (i.e., red line up) and the metal holder. Seal the plate at 180ºC for 5vs and remove the plate immediately from the sealer.
Sealed plate should have indentations around wells. Always check the sealing before loading the plate on the PCR thermal cycler.
?. List of assay primers and probes.  ABCD1CDC N1GACCCCAAAATCAGCGAAAT&nbsp;FAM-ACCCCGCATTACGTTTGGTGGACC-&nbsp;BHQ1&nbsp;TCTGGTTACTGCCAGTTGAATCTG2CDC N2TTACAAACATTGGCCGCAAAHEX-ACAATTTGCCCCCAGCGCTTCAG-IowaBHQGCGCGACATTCCGAAGAA3BCoVCTGGAAGTTGGTGGAGTTFAM&nbsp;-CCTTCATATCTATACACATCAAGTTGTT-BHQ1ATTATCGGCCTAACATACATC4HepGCGGCCAAAAGGTGGTGGATGHEX-AGGTCCCTCTGGCGCTTGTGGCGAG-&nbsp;BHQ1&nbsp;CGACGAGCCTGACGTCGGG5PMMoVGAGTGGTTTGACCTTAACGTTGAFAM-CCTACCGAAGCAAATG- BHQ1TTGTCGGTTGCAATG CAA GT
ABCD1CDC N1GACCCCAAAATCAGCGAAAT&nbsp;FAM-ACCCCGCATTACGTTTGGTGGACC-&nbsp;BHQ1&nbsp;TCTGGTTACTGCCAGTTGAATCTG2CDC N2TTACAAACATTGGCCGCAAAHEX-ACAATTTGCCCCCAGCGCTTCAG-IowaBHQGCGCGACATTCCGAAGAA3BCoVCTGGAAGTTGGTGGAGTTFAM&nbsp;-CCTTCATATCTATACACATCAAGTTGTT-BHQ1ATTATCGGCCTAACATACATC4HepGCGGCCAAAAGGTGGTGGATGHEX-AGGTCCCTCTGGCGCTTGTGGCGAG-&nbsp;BHQ1&nbsp;CGACGAGCCTGACGTCGGG5PMMoVGAGTGGTTTGACCTTAACGTTGAFAM-CCTACCGAAGCAAATG- BHQ1TTGTCGGTTGCAATG CAA GT
?. Load the plate on to a PCR thermocycler. Our lab's assay conditions are shown as below. We use Eppendorf Mastercycler Pro and the ramp speed is set to 50% for RT-ddPCR.  ABCDEFG1AssayStep 1Step 2Step 3 (40 cycles)Step 4 (40 cycles)Step 5Step 62N1/N2, BCoV, HepG50ºC 60 min95ºC 10 min94ºC 30s55ºC 1 min98ºC 10 min4ºC 30 mins and hold3PMMoV50ºC 60 min95ºC 10 min94ºC 30s60ºC 1 min98ºC 10 min4ºC 30 mins and hold
ABCDEFG1AssayStep 1Step 2Step 3 (40 cycles)Step 4 (40 cycles)Step 5Step 62N1/N2, BCoV, HepG50ºC 60 min95ºC 10 min94ºC 30s55ºC 1 min98ºC 10 min4ºC 30 mins and hold3PMMoV50ºC 60 min95ºC 10 min94ºC 30s60ºC 1 min98ºC 10 min4ºC 30 mins and hold
?. Place a 96-well PCR plate onto a low temp PCR rack, or on ice. Disperse 16.5 μL of reaction matrix into each well of a 96-well PCR plate. For runs with multiple columns, calculate (the matrix total volume/8) and evenly distribute the matrix into an 8-well PCR strip. Then use an appropriate multichannel pipette (e.g., 2- 20 μL range) to add 16.5 μL of matrix into each well, column by column.
1. Keep the PCR plate cold/on ice during the process.2. Lower the pipetting speed to avoid liquid leftover to the inner side of the tips.
?. Gently vortex at half speed to mix the RNA sample. Make sure no liquid is attached on the lid. Add 5.5 μL of sample RNA into each well containing 16.5 μL of reaction matrix, making the total volume of each reaction 22 μL.
1. Keep the PCR plate and RNA samples cold/on ice throughout the process.2. Do not over vortex the RNA samples.3. Pipetting robot may be used to add RNA samples to the matrix.
?. Prepare the reaction matrix (for one well, beside sample RNA) according to the table below. Prepare  Use a low-binding tube of appropriate volume to mix all the components according to the reaction numbers. Always include extra wells when setting up reaction to avoid potential volume shortage caused by pipetting.   ABC1ComponentVolume per reaction, uLFinal concentration2Supermix5.51x3Reverse transcriptase2.220 U/uL4300 mM DTT1.115 mM5Primer mix (forwad + reverse)1.1900 nM6Probe1.1250 nM7RNase-free water*5.5/8Total16.5/
ABC1ComponentVolume per reaction, uLFinal concentration2Supermix5.51x3Reverse transcriptase2.220 U/uL4300 mM DTT1.115 mM5Primer mix (forwad + reverse)1.1900 nM6Probe1.1250 nM7RNase-free water*5.5/8Total16.5/
1. Keep all reagents on ice during the process as well as the matrix.2. Always prepare a total of 8* (N columns) wells for droplet generation, or use ddPCR Buffer Control for Probes (#1863052) to fill empty wells on the last cartridge.3. Make sure sticky reagents are added the correct volumes and not left in the tips, i.e., Supermix, RT.
?. Seal the PCR plate. Centrifuge down gently at 1000 rpm for 30s. Take out the plate and vortex on a 96-well plate mixer at 1600 rpm for 30s. Centrifuge again at 1000 rpm for 30s to settle down the plate.
?. Prepare materials/reagents on the working bench top, e.g., cartridges, gaskets, ddPCR 96-well plate, droplet generation oil, foil cover. Label the cartridges with corresponding column numbers (e.g., 1 to 12 for a full plate).
?. Remove the sealing on the PCR plate. Place the cartridge in the cartridge holder. Align well the PCR plate and the cartridge on the bench. Use an appropriate multichannel pipette (e.g., 2- 20 μL) for liquid transfer. Adjust the pipette at 20 μL and make sure the tips are well positioned. Gently mix the liquid by aspirating up to 2/3 of the tip height and then releasing to a lower level of the tip height, i.e., not to the end of the tip, to avoid creating bubbles. Repeat this mixing step 10 times. In the last movement, slowly aspirate to the full volume of 20 μL.
?. Transfer the 20 μL reaction matrix to the middle column of the cartridge. Position the tip end to the ridge in well (where the well wall connects to the bottom) at 15º angle. Avoid creating bubbles when releasing liquid from tips into the cartridge; this can be realized by only pressing the plunger to position 2 and not position 3 before pull the tips out from the cartridge. Make sure the cartridge wells are in the same direction as on the PCR plate.
?. Fill in 70 μl of Droplet Generation Oil into bottom wells of the cartridge and cover the cartridge with a red gasket. Loop outer holes of red gasket around hooks on left and right sides of cartridge holder. Place the gasket equipped cartridge into the droplet generator.
You can start preparing the next column of droplet generation while waiting for the previous column to be done.
?. When droplet generation is done, take out the cartridge from the droplet generator and remove the red gasket. Using a Rainin multichannel with recommended Rainin tips, in a leaning position, count to 5 to aspirate all the liquid (i.e., 40 μl) from the droplets column, and press against side of wells of the corresponding column in the ddPCR 96-well plate (i.e., not the previous PCR plate), count to 5 to expel the droplets into the wells.
1. Avoid multiple times of liquid transfer.2. Eye ball the ddPCR plate when droplet generation and transferring are done.