Sep 18, 2020
COVID Airway Processing for scRNAseq
- Peter Szabo1,
- Steven Wells1,
- Peter A. Sims1,
- Donna Farber1
- 1Columbia University
- Human Cell Atlas Method Development Community
- Coronavirus Method Development Community
Protocol Citation: Peter Szabo, Steven Wells, Peter A. Sims, Donna Farber 2020. COVID Airway Processing for scRNAseq. protocols.io https://dx.doi.org/10.17504/protocols.io.bjj8kkrw
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 and it's working
Created: August 12, 2020
Last Modified: September 18, 2020
Protocol Integer ID: 40288
Keywords: SARS-CoV-2, COVID-19, lymphocytes, isolation, pan-mononuclear cells
Abstract
This protocol describes the isolation of lymphocytes and pan-mononuclear cells from airway samples.
Attachments
Materials
MATERIALS
Gibco™ DPBS no calcium no magnesiumThermo Fisher ScientificCatalog #14190144
Penicillin-Streptomycin-Glutamine (100X)Thermo FisherCatalog #10378016
UltraPure™ 0.5 M EDTA pH 8.0Thermo Fisher ScientificCatalog #15575020
Thermo Scientific™ Nunc™ 50mL Conical Sterile Polypropylene Centrifuge TubesFisher ScientificCatalog #12-565-271
5mL Falcon™ Round-Bottom Polypropylene Test TubesFisher ScientificCatalog #14-959-11A
Biotin anti-human CD235ab AntibodyBioLegendCatalog #306618
Biotin anti-human CD66b AntibodyBioLegendCatalog #305120
BioMag® Plus StreptavidinBangs LaboratoriesCatalog #BP628
Corning™ Externally Threaded Cryogenic VialsFisher ScientificCatalog #09-761-71
CryoStor CS10 100MLFisher ScientificCatalog #NC9930384
Gibco™ Fetal Bovine Serum qualified AustraliaFisher ScientificCatalog #10-099-141
Ficoll-Paque™ PLUS MediaFisher ScientificCatalog #45-001-749
Human TruStain FcX™BioLegendCatalog #422302
Gibco™ IMDM (Iscoves Modified Dulbeccos Medium)Fisher ScientificCatalog #12-440-053
NC-Slide A8™ box with 25 SlidesChemometecCatalog #942-0003
Solution 13 AO – DAPIChemometecCatalog #910-3013
Dead Cell Removal KitMiltenyi BiotecCatalog #130-090-101
MS ColumnsMiltenyi BiotecCatalog #130-042-201
Equipment
- Centrifuge
- Cell Counter - NC-3000
- EasyEights™ EasySep™ Magnet (Stemcell Technologies, Cat. No.: 18103)
Equipment
EasyEights™ EasySep™ Magnet
NAME
Magnet for column-free immunomagnetic separation
TYPE
EasySep
BRAND
18103
SKU
LINK
- MACS Multistand (Miltenyi, Cat. No.: 130-108-934)
Equipment
MACS MultiStand
NAME
Separator for magnetic cell separation
TYPE
MACS
BRAND
130-108-934
SKU
LINK
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Biosafety Notes
- All materials required for sample processing are to be prepared in the biosafety cabinet before handling of Airway samples.
- All Airway sample manipulation takes place in a biosafety cabinet unless specifically stated.
- All centrifugation steps must take place in capped containers. Upon completion of a centrifugation step, return the capped container to the biosafety cabinet, remove all tubes, and spray and wipe them with >70% ethanol before continuing to the next step.
Preparing Buffer and Media
Preparing Buffer and Media
Create the following DPBS Solution-EDTA in a bottle of DPBS by using the table below:
| Component | Volume (mL) | Starting Conc. | Final Conc. | |
| DPBS | 474 | - | ||
| FBS | 25 | 100% | 5% | |
| EDTA | 1 | 0.5 M | 1 mM |
Table 1.
Create IMDM Layering Media in a bottle of IMDM.
| Component | Volume (mL) | Starting Conc. | Final Conc. | |
| IMDM | 470 | - | ||
| FBS | 25 | 100% | 5% | |
| EDTA | 5 | 100% | 1% |
Table 2.
Preparation of Sample
Preparation of Sample
Place sample box into biosafety cabinet.
Remove samples from box and from the containment bags, discard bags, spray sample containers
with >70% ethanol and wipe down.
Note
NOTE: If sputum traps come with tubing attached flush tubing with 1 mL -2 mL (or more) before proceeding to optimize yield.
Record the total volume of Airway sample to be processed in mL.
Transfer the Airway Sample to a 50 mL tube, add 4 mL DPBS to the airway sample.
Spin the Airway for 400 x g, 20°C, 00:10:00 , remove and save four aliquots of the
supernatant in cryovials. Add the same volume to all cryovials.
Record number of vials: __________ and the volume per vial __________ mL.
Note
NOTE: Ensure that cryovials are decomtaminated prior to removal from the biosafety cabinet.
Add IMDM Layering Media to the Airway sample to bring the total volume to 25 mL .
Add 40 µL Benzonase to the sample, pipette up and down vigorously to mix and dissociate airway pellet, incubate at Room temperature for 00:30:00 .
Add 750 µL 0.5M EDTA , and filter sample through a 100 μM filter.
Ficoll-Paque
Ficoll-Paque
Aliquot 15 mL Ficoll-Paque Media PLUS to a 50 mL tube.
Using the slow setting on the pipette gun, gently layer the airway/IMDM mixture on top of the 15 mL Ficoll-Paque Media PLUS .
Take extra care not to disturb the interface while layering. Disturbing the interface excessively prevents the mononuclear cells from becoming a clean layer.
Spin for 1200 x g, 20°C, 00:20:00 with no brake, 4 acceleration.
Note
NOTE: Centrifuge should be pre-warmed to 20 °C .
Remove the mononuclear cell layer from each tube and transfer to a new 50 mL tube. Take extra care to avoid pulling cells from the ficoll layer (underneath the mononuclear cell layer) as this typically contains a lot of granulocytes. Pulling from the plasma layer is not an issue.
Top MNC with cold DPBS Solution-EDTA to 40 mL (ensure at least 2-3 volumes are added) and centrifuge the cell suspension(s) for 400 x g, 4°C, 00:10:00 .
(Platelet Spin) Discard the supernatant, top tube to 40 mL with cold DPBS Solution-EDTA, and centrifuge the cell suspension for 120 x g, 4°C, 00:10:00 .
Remove the supernatant (caution: pellet may be loose), and re-suspend the cell pellet in 4.5 mL Dulbecco’s Phosphate Buffered Saline (DPBS) (final volume should be about 5 mL ).
Cell Counting of COVID Samples
Cell Counting of COVID Samples
Add 0.05 mL sample , 0.05 mL DPBS , and 0.005 mL Solution 13 to a 1.5 mL centrifuge tube, incubate for 00:02:00 at Room temperature .
Add 0.1 mL BD Cytofix Fixation Buffer to the samples and incubate 00:30:00 , Room temperature , and protect from light.
Aliquot 0.01 mL sample to the well of a NC-Slide A8 and count on the NC-3000.
Record number and viability below, calculate total cells:
cell number: __________cells/mL, __________% viable
Division of Sample for scRNAseq Analysis and Freeze-down
Division of Sample for scRNAseq Analysis and Freeze-down
Aliquot up to 2 x 107 cells to a 5 mL Falcon Round-Bottom tube and place On ice for subsequent sample clean-up (next section).
Freeze down up to 1 x 108 cells in approximately 1 x 107 aliquots (1 mL each) using Cryostor CS10 Medium, a Mr. Frosty, and a -80 °C freezer.
Record the number of vials frozen: __________ and the cells per cryovial frozen: _________.
Sample Clean Up for scRNAseq – CD66b and CD235ab removal
Sample Clean Up for scRNAseq – CD66b and CD235ab removal
Centrifuge the single cell suspension for 400 x g, 4°C, 00:05:00 .
Discard the supernatant and resuspend the cell pellet in 50 µL DPBS Solution-EDTA .
Add 10 µL Human TruStain FcX to the single cell suspension and incubate for 00:10:00 , 4 °C .
Add 10 µL biotinylated anti-CD66b and biotinylated anti-CD235ab to the sample and incubate for 00:30:00 , 4 °C .
While the single cell suspension is incubating add 0.2 mL BioMag Plus Streptavidin Beads to a 5 mL Falcon Round-Bottom tube.
Add 2 mL DPBS Solution-EDTA to the BioMag Plus Streptavidin Beads and place on a magnet for 00:05:00 .
Remove all the supernatant from the BioMag Plus Streptavidin Beads, remove from the magnet and resuspend the beads in 0.1 mL DPBS Solution-EDTA .
Once step 26 (go to step #26 ) is complete, add 3 mL DPBS Solution-EDTA to the single cell suspension and centrifuge for 400 x g, 4°C, 00:05:00 .
Resuspend the single cell suspension in the BioMag Plus Streptavidin Beads from step 29 (go to step #29 ), and incubate at Room temperature for 00:05:00 .
Add 3 mL DPBS to the tube and place on a magnet for 00:05:00 .
Remove supernatant from tube and transfer to a separate 5 mL Falcon Round Bottom tube.
Sample Clean Up for scRNAseq – Dead Cell Removal
Sample Clean Up for scRNAseq – Dead Cell Removal
Centrifuge the single cell suspension for 400 x g, 4°C, 00:05:00 , and discard supernatant.
Resuspend cell pellet in 0.1 mL Dead Cell Removal Microbeads , mix well, incubate, Room temperature , 00:15:00 .
While the cell suspension is incubating, place an MS Column onto the MACS Multistand and rinse with 0.5 mL 1x Binding Buffer Solution .
Post incubation, apply cell suspension to the MS Column and capture the flow through in a 5 mL Falcon Round Bottom tube.
Rinse with 1.5 mL 1x Binding Buffer and capture in the same tube.
Centrifuge the single cell suspension for 400 x g, 4°C, 00:05:00 , and discard supernatant.
Resuspend cell pellet in 0.5 mL DPBS , and count cells.
Cell Counting of COVID Samples (10x)
Cell Counting of COVID Samples (10x)
Add 0.05 mL sample , 0.05 mL DPBS , and 0.005 mL Solution 13 to a 1.5 mL centrifuge tube, incubate for 00:02:00 at Room temperature .
Add 0.1 mL BD Cytofix Fixation Buffer to the samples and incubate 00:30:00 , Room temperature , and protect from light.
Aliquot 0.01 mL sample to the well of a NC-Slide A8 and count on the NC-3000.
Record number and viability below, calculate total cells:
cell number: __________cells/mL, __________% viable
10X Encapsulation
10X Encapsulation
Follow the appropriate 10X protocol (Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 User Guide – Rev D) for encapsulation of cells from the airway sample.