TITLE: Microwell-based Single-Cell RNA-seq
AUTHORS: Peter Sims, Jinzhou Yuan, Yim L. Cheng

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">(</span><span style = "font-weight:bold;">On</span><span>-chip reverse transcription version, Sims Lab – Jinzhou Yuan and Yim L. Cheng) 7/6/2017</span></div></div>

[STEPS]
?. [Experiment day 1]
Live stain cellsHarvest and resuspend 500,000 cells in (500 cells/uL).
[TBS]
?. [Experiment day 1]
Incubate the cells in 4 uM CalceinAM (4ul 1mM CalceinAM per ml cells) for  on ice.
?. [Experiment day 1]
Preparation of lysis buffer, and wash buffer, and fluorinated oilWhile the cells are being live stained, make ( + ) ( + ).
[lysis buffer]
[buffer TCL]
[2-Mercaptoethanol]
[RNase inhibitor-doped wash buffer]
[wash buffer]
[SUPERaseIN]
?. [Experiment day 1]
Load buffers and fluorinated oil to reagent reservoirs  reagent reservoir 1:  reagent reservoir 4:  reagent reservoir 5:
[lysis buffer]
[fluorinated oil ]
[RNase inhibitor-doped wash buffer ]
?. [Experiment day 1]
Prepare mRNA-capture-beadsGently vortex the beads, transfer  of beads into a microcentrifuge tube.
500 µl
?. [Experiment day 1]
Wash the beads with and resuspend the beads in . Keep the beads on ice before use.
[TBS buffer]
[TBS buffer]
?. [Experiment day 1]
mRNA-capture-bead and cell loadingsFlush  through the wash buffer-filled device. Exercise caution to avoid flowing air into the device.
[TBS]
?. [Experiment day 1]
Load  into the TBS-flushed device. Fine tune the size of liquid puddles on both inlet and outlet of the device until the cells stop moving in the device. Let cells settle for .
[live-stained cells]
?. [Experiment day 1]
Flush  through the cell-loaded device. Flush gently so that the loaded cells don’t get washed out.
[TBS]
?. [Experiment day 1]
Confirm 5-10% of wells contain single cell under microscope.
?. [Experiment day 1]
Load into the device. Reuse the beads to repeat the loading until >60% wells contain beads. Load gently so that the bead flow does not wash cells out.
[TBS-washed beads]
?. [Experiment day 1]
Flush  through the cell-bead-loaded device. Flush gently so that the loaded cells and beads don’t get washed out.
[TBS]
?. [Experiment day 1]
Confirm >60% of wells contain mRNA-capture-bead under microscope.
?. [Experiment day 1]
Pre-run fluidics wash Open channel 1 for  by executing the following command in the MinGW terminal: “Valvesonly_3_dual_3.exe 10000 1”.
?. [Experiment day 1]
Open channel 4 for  by executing the following command in the MinGW terminal: “Valvesonly_3_dual_3.exe 10000 4”.
?. [Experiment day 1]
Open channel 5 for  by executing the following command in the MinGW terminal: “Valvesonly_3_dual_3.exe 30000 5”.
?. [Experiment day 1]
Connect the device to the fluidics system and start the runPlug the inflow (red) and outflow (green) tubing to the inlet and outlet of the device respectively. Make sure that the tubings are NOT inserted all the way to the bottom of the inlet or outlet as this can clog the device, which will prevent liquid from flowing through or flow at a significantly reduced flow rate.
?. [Experiment day 1]
Start the run by executing the following commend in the MinGW terminal: “SingleCellRNACapture_TCL_dual_large_array_3.exe”. Lysis buffer will be pushed through the device immediately followed by fluorinated oil, which will seal the wells, physically isolating the lysate of each individual cell.
?. [Experiment day 1]
The device will be heated to  to promote cell lysis and mRNA capture, and then cooled back to room temperature.
?. [Experiment day 1]
Cell lysis QC and mRNA captureOnce the device has been cooled to room temperature, disconnect the device from the fluidics system. Seal both inlet and outlet of the device with disc tapes.
?. [Experiment day 1]
Imaging the device under fluorescence microscope with a 10X objective. Check the distribution of lysate (traced by the live stain dye) in the device. You should see the fluorescent dye fill up the wells that had cells, and the absence of fluorescent dye in wells that did not have cell. If this is the case, unseal the inlet and outlet of the device, reconnect the device to the fluidics system, and proceed to the next step. Otherwise, abort the experiment.
?. [Experiment day 1]
Reverse transcriptionPrepare the following reverse transcription mixture  ABC1Final ConcentrationVolumeStock Concentration21X Maxima RT buffer100 µL5X Maxima RT buffer31 mM dNTPs50 µL10 mM dNTP41 U/ul SuperaseIN25 µL20 U/ul SuperaseIN52.5 uM SMRT_TSO12.5 µL100 uM SMRT_TSO610 U/ul Maxima H- RT25 µL200 U/ul Maxima H- RT70.1% Tween 205 µL10% Tween208Nuclease free water282.5 µL9Total500 µL
ABC1Final ConcentrationVolumeStock Concentration21X Maxima RT buffer100 µL5X Maxima RT buffer31 mM dNTPs50 µL10 mM dNTP41 U/ul SuperaseIN25 µL20 U/ul SuperaseIN52.5 uM SMRT_TSO12.5 µL100 uM SMRT_TSO610 U/ul Maxima H- RT25 µL200 U/ul Maxima H- RT70.1% Tween 205 µL10% Tween208Nuclease free water282.5 µL9Total500 µL
?. [Experiment day 1]
Load all  to reagent reservoir 2 before mRNA capture step is completed.
[reverse transcription mixture]
?. [Experiment day 1]
Bead extractionAssemble a syringe-tubing set: 2.5 cm green PEEK tubing and 1ml syringe connected with 12 cm tygon tubing.
?. [Experiment day 1]
Prepare , keep on ice
[wash buffer]
?. [Experiment day 1]
“Massage” the device to dislodge the beads from the device, then flow  through the device with syringe-tubing set, COLLECT the overflow.
[wash buffer]
cDNA-coated beads will be washed out from the device with the buffer flow, so you want to collect the overflow.
?. [Experiment day 1]
Repeat step 26 until >95% of the beads have been removed from the device.
[Repeat step 26]
?. [Experiment day 1]
Wash the cDNA-beads with (10 mM Tris pH 8.0, 1 mM EDTA, 0.5% SDS)
[TE/SDS buffer]
?. [Experiment day 1]
Resuspend the cDNA-beads in .
[TE/TW buffer]
?. [Experiment day 1]
Store cDNA-beads at .
?. [Experiment day 1]
Wash the cDNA-beads with (10 mM Tris pH 8.0, 1 mM EDTA, 0.01% Tween-20) (1/2)
[TE/TW buffer]
?. [Experiment day 1]
Wash the cDNA-beads with (10 mM Tris pH 8.0, 1 mM EDTA, 0.01% Tween-20) (2/2)
[TE/TW buffer]
?. [Experiment day 1]
Wash fluidics systemRinse and fill ALL reagent reservoirs (except reservoir 4) with DI water.
?. [Experiment day 1]
Execute the following command in the MinGW terminal: “SingleCellRNACapture_wash_dual_3.exe”
?. [Experiment day 2]
Exo-I reactionPrepare the following Exo-I reaction mixture  ABC1Final ConcentrationVolumeStock Concentration21X Exo I buffer5 µL10X Exo I buffer31 U/uL Exo I2.5 µL20 U/uL4Nuclease free water42.5 µL5Total50 µL
ABC1Final ConcentrationVolumeStock Concentration21X Exo I buffer5 µL10X Exo I buffer31 U/uL Exo I2.5 µL20 U/uL4Nuclease free water42.5 µL5Total50 µL
?. [Experiment day 2]
Wash the cDNA-beads with . (1/2)
[nuclease-free water]
?. [Experiment day 2]
Wash the cDNA-beads with . (2/2)
[nuclease-free water]
?. [Experiment day 2]
Resuspend the beads in .
[Exo-I reaction mixture]
?. [Experiment day 2]
Run the following program on a thermocycler:  ABC1TemperatureTimeNumber of cycles237 °C30 minutes134 °CHold
ABC1TemperatureTimeNumber of cycles237 °C30 minutes134 °CHold
?. [Experiment day 2]
Wash the ExoI-treated cDNA-beads with .
[TE/SDS buffer]
?. [Experiment day 2]
Wash the ExoI-treated cDNA-beads with . (1/2)
[TE/TW buffer]
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Wash the ExoI-treated cDNA-beads with . (1/2)
[nuclease-free water]
?. [Experiment day 2]
Wash the ExoI-treated cDNA-beads with . (2/2)
[TE/TW buffer]
?. [Experiment day 2]
Wash the ExoI-treated cDNA-beads with . (2/2)
[nuclease-free water]
?. [Experiment day 2]
SMRT PCR reaction (cDNA PCR amplification)Prepare the following SMRT PCR reaction mixture  ABC1Final ConcentrationVolumeStock Concentration21X PCR Ready Mix500 µL2X PCR Ready Mix31 uM SMRT PCR primer10 µL100 uM SMRT PCR primer4Nuclease free water490 µL5Total1000 µL
ABC1Final ConcentrationVolumeStock Concentration21X PCR Ready Mix500 µL2X PCR Ready Mix31 uM SMRT PCR primer10 µL100 uM SMRT PCR primer4Nuclease free water490 µL5Total1000 µL
?. [Experiment day 2]
Resuspend cDNA-beads in . Aliquot  per PCR tube.
[SMRT PCR reaction mixture]
50 µl
?. [Experiment day 2]
Run the following PCR program on a thermocycler:
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SMRT PCR product purification with 0.6X Ampure beadsCombine all SMRT PCR products into a single microcentrifuge tube.
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Spin for , transfer  to a new microcentrifuge tube.
[supernatant]
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Add  (0.6X volume) to tube. Incubate at room temperature for .
[Ampure beads]
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Place tube on a magnetic stand for . Discard the supernatant.
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Wash Ampure beads with 80% ethanol twiceAdd  to tube, wait for , remove 80% ethanol. Quick spin for , place it back on a magnetic stand for , and remove residual 80% ethanol.
[freshly-made 80% ethanol in nuclease-free water]
?. [Experiment day 2]
Cover the tube with a KimWipe, air dry the Ampure beads on the magnetic stand for .
?. [Experiment day 2]
Elute cDNA off Ampure beads with 20 ul nuclease-free waterAdd  directly on the Ampure beads, remove the tube from the magnetic stand, mix by pipetting. Incubate at room temperature for .Place tube on a magnetic stand for .
[nuclease-free water]
?. [Experiment day 2]
Store cDNA at .
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cDNA QC with Qubit and Bioanalyzer (follow vendor’s exact instructions).
?. [Experiment day 2]
Nextera tagmentation reaction (Nextera XT kit)Add  to a PCR tube.
[TD Buffer]
?. [Experiment day 2]
Add  of 0.6 ng cDNA plus nuclease free water.
5 µl
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Add  to the PCR tube. Mix by pipetting.
[ATM]
?. [Experiment day 2]
Run the following program on a thermocycler:  ABC1TemperatureTimeNumber of cycles255 °C5 minutes1310 °Chold
ABC1TemperatureTimeNumber of cycles255 °C5 minutes1310 °Chold
?. [Experiment day 2]
Immediately after the temperature reaches 10 ºC, add  to the PCR tube and mix by pipetting.
[NT Buffer]
?. [Experiment day 2]
Incubate at room temperature for .
?. [Experiment day 2]
Selective amplification of the 3' end fragment of cDNA (Nextera XT kit)Add the following reagents to the end product in step 62 and mix by pipetting:  AB1ReagentVolume2NPM15 µL3N7 PCR primer5 µL42 uM Custom P5 primer (not from Nextera XT kit)5 µL
AB1ReagentVolume2NPM15 µL3N7 PCR primer5 µL42 uM Custom P5 primer (not from Nextera XT kit)5 µL
?. [Experiment day 2]
Run the following program on a thermocycler:
?. [Experiment day 2]
Nextera PCR product purification with 0.6X + 1X Ampure beadQuick spin the Nextera PCR product , transfer  to a new microcentrifuge tube.
[PCR product]
?. [Experiment day 2]
Add (0.6X volume) to tube. Incubate at room temperature for .
[Ampure beads]
?. [Experiment day 2]
Place tube on a magnetic stand for . Discard the supernatant.
?. [Experiment day 2]
Wash Ampure beads with 80% ethanol twiceAdd  to tube, wait for , remove 80% ethanol.Quick spin for , place it back on a magnetic stand for , and remove residual 80% ethanol.
[freshly-made 80% ethanol in nuclease-free water]
?. [Experiment day 2]
Cover the tube with a KimWipe, air dry the Ampure beads on the magnetic stand for .
?. [Experiment day 2]
Elute the Nextera library off Ampure beads with 50 ul nuclease-free waterAdd directly on the Ampure beads, remove the tube from the magnetic stand, mix the beads and the water by pipetting. Incubate at room temperature for .Place tube on a magnetic stand for .
[nuclease-free water]
?. [Experiment day 2]
Repeat Ampure bead purification steps (steps 65-70) with 1x volume Ampure bead (50 ul) and elute the Nextera library off Ampure beads with .
[nuclease-free water]
?. [Experiment day 2]
Store the Nextera library at .
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Nextera library QC with Qubit and Bioanalyzer (follow vendor’s exact instructions).
?. [Experiment day 2]
Sequencing Use custom read 1 sequencing primer.Use 20% phix.21 cycles on read 1.63 cycles on read 2.8 cycles on index read 1 (if multiple samples are pooled together).