Apr 26, 2019

Public workspaceMicrowell-based Single-Cell RNA-seq

DOI
dx.doi.org/10.17504/protocols.io.yqmfvu6
  • 1Columbia University Irving Medical Center
  • Human Cell Atlas Method Development Community
Peter Sims
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DOI: dx.doi.org/10.17504/protocols.io.yqmfvu6
Protocol Citation: Peter Sims, Jinzhou Yuan, Yim L. Cheng 2019. Microwell-based Single-Cell RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.yqmfvu6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol in our group and it is working.
Created: March 01, 2019
Last Modified: April 26, 2019
Protocol Integer ID: 20973
Keywords: single-cell, RNA-seq
Abstract
(On-chip reverse transcription version, Sims Lab – Jinzhou Yuan and Yim L. Cheng) 7/6/2017
Attachments
Icon for Microfluidic Single-Cell RNA-seq-On-chip-reverse-transcription-Sims_Lab-YLC_clean.docx
Microfluidic Single-...
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Guidelines
Our single-cell RNA-seq technology uses a custom microfluidic device that we produce in our lab.

The CAD drawings of these devices that one can submit to a foundry for photolithographic production of molds from which these devices can be routinely generated:

http://www.columbia.edu/~pas2182/localhost/protocols/First_Mold_JY_CU_6inchwafer_microwellarrays_50umthick.dwg

http://www.columbia.edu/~pas2182/localhost/protocols/Second_Mold_JY_CU_6inchwafer_flowcell_100umthick.dwg

Safety warnings
Please refer to the SDS (Safety Data Sheet) for information about hazards, and to obtain advice on safety precautions.
Before start
Day before experiment:

Device Preparation
  • Fill a new device with wash buffer (20mM Tris-HCl pH7.9, 50mM NaCl, 0.1% Tween 20). Cover both the inlet and outlet of the device with a puddle of wash buffer.
  • Store the wash buffer filled device in a humid chamber (P1000 pipette tip container filled with water) at room temperature.
Experiment day 1
Experiment day 1
Live stain cells

Harvest and resuspend 500,000 cells in Amount1 mL TBS (500 cells/uL).


Incubate the cells in 4 uM CalceinAM (4ul 1mM CalceinAM per ml cells) for Duration00:15:00 on ice.

Preparation of lysis buffer, and wash buffer, and fluorinated oil

While the cells are being live stained, make
Amount1 mL lysis buffer (Amount990 µL buffer TCL + Amount10 µL 2-Mercaptoethanol )
Amount2 mL RNase inhibitor-doped wash buffer (Amount1998 µL wash buffer + Amount2 µL SUPERaseIN ).
Load buffers and fluorinated oil to reagent reservoirs
  • reagent reservoir 1: Amount1 mL lysis buffer
  • reagent reservoir 4: Amount1.8 mL fluorinated oil
  • reagent reservoir 5: Amount2 mL RNase inhibitor-doped wash buffer
Prepare mRNA-capture-beads

Gently vortex the beads, transfer Amount500 µL of beads into a microcentrifuge tube.
Wash the beads with Amount1 mL TBS buffer and resuspend the beads in Amount400 µL TBS buffer . Keep the beads on ice before use.


mRNA-capture-bead and cell loadings

Flush Amount1 mL TBS through the wash buffer-filled device. Exercise caution to avoid flowing air into the device.

Load Amount500 µL live-stained cells 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 Duration00:03:00 .
Flush Amount1 mL TBS through the cell-loaded device. Flush gently so that the loaded cells don’t get washed out.
Confirm 5-10% of wells contain single cell under microscope.
Load Amount400 µL TBS-washed beads 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.

Flush Amount1 mL TBS through the cell-bead-loaded device. Flush gently so that the loaded cells and beads don’t get washed out.

Confirm >60% of wells contain mRNA-capture-bead under microscope.
Pre-run fluidics wash

Open channel 1 for Duration00:00:10 by executing the following command in the MinGW terminal: “Valvesonly_3_dual_3.exe 10000 1”.


Open channel 4 for Duration00:00:10 by executing the following command in the MinGW terminal: “Valvesonly_3_dual_3.exe 10000 4”.


Open channel 5 for Duration00:00:30 by executing the following command in the MinGW terminal: “Valvesonly_3_dual_3.exe 30000 5”.


Connect the device to the fluidics system and start the run

Plug 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.
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.
The device will be heated to Temperature50 °C to promote cell lysis and mRNA capture, and then cooled back to room temperature.

Cell lysis QC and mRNA capture

Once 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.
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.
Reverse transcription

Prepare the following reverse transcription mixture
Final ConcentrationVolumeStock Concentration
1X Maxima RT buffer100 µL5X Maxima RT buffer
1 mM dNTPs50 µL10 mM dNTP
1 U/ul SuperaseIN25 µL20 U/ul SuperaseIN
2.5 uM SMRT_TSO12.5 µL100 uM SMRT_TSO
10 U/ul Maxima H- RT25 µL200 U/ul Maxima H- RT
0.1% Tween 205 µL10% Tween20
Nuclease free water282.5 µL
Total500 µL

Load all Amount500 µL reverse transcription mixture to reagent reservoir 2 before mRNA capture step is completed.

Bead extraction

Assemble a syringe-tubing set: 2.5 cm green PEEK tubing and 1ml syringe connected with 12 cm tygon tubing.
Prepare Amount3 mL wash buffer , keep on ice

“Massage” the device to dislodge the beads from the device, then flow Amount1 mL wash buffer through the device with syringe-tubing set, COLLECT the overflow.

Note
cDNA-coated beads will be washed out from the device with the buffer flow, so you want to collect the overflow.

Repeat step 26 until >95% of the beads have been removed from the device.
Go to Repeat step 26
Wash the cDNA-beads with

Amount1 mL TE/SDS buffer (10 mM Tris pH 8.0, 1 mM EDTA, 0.5% SDS)


Wash the cDNA-beads with

Amount1 mL TE/TW buffer (10 mM Tris pH 8.0, 1 mM EDTA, 0.01% Tween-20) (1/2)

Wash the cDNA-beads with

Amount1 mL TE/TW buffer (10 mM Tris pH 8.0, 1 mM EDTA, 0.01% Tween-20) (2/2)

Resuspend the cDNA-beads in Amount1 mL TE/TW buffer .

Store cDNA-beads at Temperature4 °C .
Wash fluidics system

Rinse and fill ALL reagent reservoirs (except reservoir 4) with DI water.
Execute the following command in the MinGW terminal: “SingleCellRNACapture_wash_dual_3.exe”
Experiment day 2
Experiment day 2
Exo-I reaction

Prepare the following Exo-I reaction mixture
Final ConcentrationVolumeStock Concentration
1X Exo I buffer5 µL10X Exo I buffer
1 U/uL Exo I2.5 µL20 U/uL
Nuclease free water42.5 µL
Total50 µL

Wash the cDNA-beads with Amount1 mL nuclease-free water . (1/2)

Wash the cDNA-beads with Amount1 mL nuclease-free water . (2/2)
Resuspend the beads in Amount50 µL Exo-I reaction mixture .

Run the following program on a thermocycler:
TemperatureTimeNumber of cycles
37 °C30 minutes1
4 °CHold

Wash the ExoI-treated cDNA-beads with Amount1 mL TE/SDS buffer .

Wash the ExoI-treated cDNA-beads with Amount1 mL TE/TW buffer . (1/2)


Wash the ExoI-treated cDNA-beads with Amount1 mL TE/TW buffer . (2/2)
Wash the ExoI-treated cDNA-beads with Amount1 mL nuclease-free water . (1/2)

Wash the ExoI-treated cDNA-beads with Amount1 mL nuclease-free water . (2/2)
SMRT PCR reaction (cDNA PCR amplification)

Prepare the following SMRT PCR reaction mixture
Final ConcentrationVolumeStock Concentration
1X PCR Ready Mix500 µL2X PCR Ready Mix
1 uM SMRT PCR primer10 µL100 uM SMRT PCR primer
Nuclease free water490 µL
Total1000 µL

Resuspend cDNA-beads in Amount1000 µL SMRT PCR reaction mixture . Aliquot Amount50 µL per PCR tube.
Run the following PCR program on a thermocycler:



SMRT PCR product purification with 0.6X Ampure beads

Combine all SMRT PCR products into a single microcentrifuge tube.
Spin for Duration00:01:00 , transfer Amount900 µL supernatant to a new microcentrifuge tube.

Add Amount540 µL Ampure beads (0.6X volume) to tube. Incubate at room temperature for Duration00:08:00 .

Place tube on a magnetic stand for Duration00:05:00 . Discard the supernatant.

Wash Ampure beads with 80% ethanol twice
  • Add Amount1 mL freshly-made 80% ethanol in nuclease-free water to tube, wait for Duration00:00:30 , remove 80% ethanol.
  • Repeat 80% ethanol wash.
  • Quick spin for Duration00:00:30 , place it back on a magnetic stand for Duration00:00:30 , and remove residual 80% ethanol.
Cover the tube with a KimWipe, air dry the Ampure beads on the magnetic stand for Duration00:05:00 .

Elute cDNA off Ampure beads with 20 ul nuclease-free water
  • Add Amount20 µL nuclease-free water directly on the Ampure beads, remove the tube from the magnetic stand, mix by pipetting.
  • Incubate at room temperature for Duration00:05:00 .
  • Place tube on a magnetic stand for Duration00:03:00 .
  • Keep supernatant (amplified cDNA).
Store cDNA at Temperature-20 °C .

cDNA QC with Qubit and Bioanalyzer (follow vendor’s exact instructions).
Nextera tagmentation reaction (Nextera XT kit)

Add Amount10 µL TD Buffer to a PCR tube.
Add Amount5 µL of 0.6 ng cDNA plus nuclease free water.
Add Amount5 µL ATM to the PCR tube. Mix by pipetting.
Run the following program on a thermocycler:
TemperatureTimeNumber of cycles
55 °C5 minutes1
10 °Chold

Immediately after the temperature reaches 10 ºC, add Amount5 µL NT Buffer to the PCR tube and mix by pipetting.
Incubate at room temperature for Duration00:05:00 .
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:
ReagentVolume
NPM15 µL
N7 PCR primer5 µL
2 uM Custom P5 primer (not from Nextera XT kit)5 µL

Run the following program on a thermocycler:



Nextera PCR product purification with 0.6X + 1X Ampure bead

Quick spin the Nextera PCR product Duration00:01:00 , transfer Amount50 µL PCR product to a new microcentrifuge tube.
Add Amount30 µL Ampure beads (0.6X volume) to tube. Incubate at room temperature for Duration00:08:00 .


Place tube on a magnetic stand for Duration00:05:00 . Discard the supernatant.
Wash Ampure beads with 80% ethanol twice
  • Add Amount200 µL freshly-made 80% ethanol in nuclease-free water to tube, wait for Duration00:00:30 , remove 80% ethanol.
  • Repeat 80% ethanol wash.
  • Quick spin for Duration00:00:30 , place it back on a magnetic stand for Duration00:00:30 , and remove residual 80% ethanol.
Cover the tube with a KimWipe, air dry the Ampure beads on the magnetic stand for Duration00:05:00 .
Elute the Nextera library off Ampure beads with 50 ul nuclease-free water
  • Add Amount50 µL nuclease-free water 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 Duration00:05:00 .
  • Place tube on a magnetic stand for Duration00:03:00 .
  • Keep supernatant (amplified cDNA).
Repeat Ampure bead purification steps (steps 65-70) with 1x volume Ampure bead (50 ul) and elute the Nextera library off Ampure beads with Amount20 µL nuclease-free water .
Go to
Store the Nextera library at Temperature-20 °C .
Nextera library QC with Qubit and Bioanalyzer (follow vendor’s exact instructions).
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).