TITLE: COVID Airway Processing for scRNAseq
AUTHORS: Peter Szabo, Steven Wells, Peter A. Sims, Donna Farber

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the isolation of lymphocytes and pan-mononuclear cells from airway samples.</div></div>

[STEPS]
?. [Preparing Buffer and Media]
Create the following DPBS Solution-EDTA in a bottle of DPBS by using the table below:  ABCD1ComponentVolume (mL)Starting Conc.Final Conc.2DPBS474-3FBS25100%5%4EDTA10.5 M1 mM
ABCD1ComponentVolume (mL)Starting Conc.Final Conc.2DPBS474-3FBS25100%5%4EDTA10.5 M1 mM
?. [Preparing Buffer and Media]
Create IMDM Layering Media in a bottle of IMDM.  ABCD1ComponentVolume (mL)Starting Conc.Final Conc.2IMDM470-3FBS25100%5%4EDTA5100%1%
ABCD1ComponentVolume (mL)Starting Conc.Final Conc.2IMDM470-3FBS25100%5%4EDTA5100%1%
?. [Preparation of Sample]
Place sample box into biosafety cabinet.
?. [Preparation of Sample]
Remove samples from box and from the containment bags, discard bags, spray sample containerswith >70% ethanol and wipe down.
NOTE: If sputum traps come with tubing attached flush tubing with  -  (or more) before proceeding to optimize yield.
?. [Preparation of Sample]
Record the total volume of Airway sample to be processed in mL.
?. [Preparation of Sample]
Transfer the Airway Sample to a 50 mL tube, add  to the airway sample.
[DPBS]
?. [Preparation of Sample]
Spin the Airway for , remove and save four aliquots of thesupernatant in cryovials. Add the same volume to all cryovials.Record number of vials: __________ and the volume per vial __________ mL.
Centrifuge: 400 34, 10 min, 20 10
NOTE: Ensure that cryovials are decomtaminated prior to removal from the biosafety cabinet.
?. [Preparation of Sample]
Add IMDM Layering Media to the Airway sample to bring the total volume to .
25 mL
?. [Preparation of Sample]
Add  to the sample, pipette up and down vigorously to mix and dissociate airway pellet, incubate at  for .
[Benzonase]
0 Room temperature
?. [Preparation of Sample]
Add , and filter sample through a 100 μM filter.
[0.5M EDTA]
?. [Ficoll-Paque]
Aliquot  to a 50 mL tube.
[Ficoll-Paque Media PLUS]
?. [Ficoll-Paque]
Using the slow setting on the pipette gun, gently layer the airway/IMDM mixture on top of the . Take extra care not to disturb the interface while layering. Disturbing the interface excessively prevents the mononuclear cells from becoming a clean layer.
[Ficoll-Paque Media PLUS]
?. [Ficoll-Paque]
Spin for  with no brake, 4 acceleration.
Centrifuge: 1200 34, 20 min, 20 10
NOTE: Centrifuge should be pre-warmed to  .
?. [Ficoll-Paque]
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.
?. [Ficoll-Paque]
Top MNC with cold DPBS Solution-EDTA to  (ensure at least 2-3 volumes are added) and centrifuge the cell suspension(s) for .
40 mL
Centrifuge: 400 34, 10 min, 4 10
?. [Ficoll-Paque]
(Platelet Spin) Discard the supernatant, top tube to  with cold DPBS Solution-EDTA, and centrifuge the cell suspension for .
40 mL
Centrifuge: 120 34, 10 min, 4 10
?. [Ficoll-Paque]
Remove the supernatant (caution: pellet may be loose), and re-suspend the cell pellet in  (final volume should be about ).
[Dulbecco’s Phosphate Buffered Saline (DPBS)]
5 mL
?. [Cell Counting of COVID Samples]
Add , , and  to a 1.5 mL centrifuge tube, incubate for  at .
[sample]
[DPBS]
[Solution 13]
0 Room temperature
?. [Cell Counting of COVID Samples]
Add  to the samples and incubate , , and protect from light.
[BD Cytofix Fixation Buffer]
0 Room temperature
?. [Cell Counting of COVID Samples]
Aliquot  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
[sample]
?. [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  for subsequent sample clean-up (next section).
on ice
?. [Division of Sample for scRNAseq Analysis and Freeze-down]
Freeze down up to 1 x 108 cells in approximately 1 x 107 aliquots ( each) using Cryostor CS10 Medium, a Mr. Frosty, and a  freezer. Record the number of vials frozen: __________ and the cells per cryovial frozen: _________.
1 mL
-80 °C
?. [Sample Clean Up for scRNAseq – CD66b and CD235ab removal]
Centrifuge the single cell suspension for .
Centrifuge: 400 34, 5 min, 4 10
?. [Sample Clean Up for scRNAseq – CD66b and CD235ab removal]
Discard the supernatant and resuspend the cell pellet in .
[DPBS Solution-EDTA]
?. [Sample Clean Up for scRNAseq – CD66b and CD235ab removal]
Add  to the single cell suspension and incubate for , .
[Human TruStain FcX]
4 °C
?. [Sample Clean Up for scRNAseq – CD66b and CD235ab removal]
Add  to the sample and incubate for , .
[biotinylated anti-CD66b and biotinylated anti-CD235ab]
4 °C
?. [Sample Clean Up for scRNAseq – CD66b and CD235ab removal]
While the single cell suspension is incubating add  to a 5 mL Falcon Round-Bottom tube.
[BioMag Plus Streptavidin Beads]
?. [Sample Clean Up for scRNAseq – CD66b and CD235ab removal]
Add  to the BioMag Plus Streptavidin Beads and place on a magnet for .
[DPBS Solution-EDTA]
?. [Sample Clean Up for scRNAseq – CD66b and CD235ab removal]
Remove all the supernatant from the BioMag Plus Streptavidin Beads, remove from the magnet and resuspend the beads in .
[DPBS Solution-EDTA]
?. [Sample Clean Up for scRNAseq – CD66b and CD235ab removal]
Once step 26 () is complete, add  to the single cell suspension and centrifuge for .
[DPBS Solution-EDTA]
Centrifuge: 400 34, 5 min, 4 10
?. [Sample Clean Up for scRNAseq – CD66b and CD235ab removal]
Resuspend the single cell suspension in the BioMag Plus Streptavidin Beads from step 29 (), and incubate at  for .
0 Room temperature
?. [Sample Clean Up for scRNAseq – CD66b and CD235ab removal]
Add  to the tube and place on a magnet for .
[DPBS]
?. [Sample Clean Up for scRNAseq – CD66b and CD235ab removal]
Remove supernatant from tube and transfer to a separate 5 mL Falcon Round Bottom tube.
?. [Sample Clean Up for scRNAseq – Dead Cell Removal]
Centrifuge the single cell suspension for , and discard supernatant.
Centrifuge: 400 34, 5 min, 4 10
?. [Sample Clean Up for scRNAseq – Dead Cell Removal]
Resuspend cell pellet in , mix well, incubate, , .
[Dead Cell Removal Microbeads]
0 Room temperature
?. [Sample Clean Up for scRNAseq – Dead Cell Removal]
While the cell suspension is incubating, place an MS Column onto the MACS Multistand and rinse with .
[1x Binding Buffer Solution]
?. [Sample Clean Up for scRNAseq – Dead Cell Removal]
Post incubation, apply cell suspension to the MS Column and capture the flow through in a 5 mL Falcon Round Bottom tube.
?. [Sample Clean Up for scRNAseq – Dead Cell Removal]
Rinse with  and capture in the same tube.
[1x Binding Buffer]
?. [Sample Clean Up for scRNAseq – Dead Cell Removal]
Centrifuge the single cell suspension for , and discard supernatant.
Centrifuge: 400 34, 5 min, 4 10
?. [Sample Clean Up for scRNAseq – Dead Cell Removal]
Resuspend cell pellet in , and count cells.
[DPBS]
?. [Cell Counting of COVID Samples (10x)]
Add , , and  to a 1.5 mL centrifuge tube, incubate for  at .
[sample]
[DPBS]
[Solution 13]
0 Room temperature
?. [Cell Counting of COVID Samples (10x)]
Add  to the samples and incubate , , and protect from light.
[BD Cytofix Fixation Buffer]
0 Room temperature
?. [Cell Counting of COVID Samples (10x)]
Aliquot  to the well of a NC-Slide A8 and count on the NC-3000.
[sample]
?. [Cell Counting of COVID Samples (10x)]
Record number and viability below, calculate total cells: cell number: __________cells/mL, __________% viable
?. [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.