TITLE: COVID Blood 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 human whole blood for scRNAseq analysis.</div></div>

[STEPS]
?. [Preparing Buffer]
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
?. [Preparation of Blood]
Place sample box into biosafety cabinet.
?. [Preparation of Blood]
Remove samples from the box and from the containment bags, discard bags, spray sample containers with >70% ethanol and wipe down.
?. [Preparation of Blood]
Record the total volume of whole blood to be processed in mL.
?. [Preparation of Blood]
Spin the whole blood  in the anti-coagulant tubes and remove the plasma layer to cryovials. Be sure to not remove any red blood cells with the plasma. 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 Blood]
Add the same volume of DPBS Solution-EDTA as removed plasma back to the blood tube. Pipette to mix.
?. [RosetteSep and Ficoll-Paque]
Add  directly to the blood tube. Pipette to mix.
[RosetteSep Cocktail/1mL of blood]
?. [RosetteSep and Ficoll-Paque]
Inclubate samples at  for .
0 Room temperature
?. [RosetteSep and Ficoll-Paque]
Transfer sample to a 50 mL tube and dilute up to  with DPBS Solution-EDTA solution.
25 mL
?. [RosetteSep and Ficoll-Paque]
Aliquot  to a 50 mL tube.
[Ficoll-Paque Media PLUS]
?. [RosetteSep and Ficoll-Paque]
Using the slow setting on the pipette gun, gently layer the blood/DPBS Solution-EDTA mixture on top of the . Take extra care not to disturb the blood-ficoll interface while layering. Disturbing the interface excessively prevents the mononuclear cells from becoming a clean layer.
[Ficoll-Paque Media PLUS]
?. [RosetteSep and Ficoll-Paque]
Spin for  with no brake, 4 acceleration.
Centrifuge: 1200 34, 20 min, 20 10
NOTE: Centrifuge should be pre-warmed to  .
?. [RosetteSep and 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.
?. [RosetteSep and Ficoll-Paque]
Top PBMC 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
?. [RosetteSep and 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
?. [RosetteSep and Ficoll-Paque]
Remove the supernatant (caution: pellet may be loose), and resuspend the cell pellet in .
[Dulbecco’s Phosphate Buffered Saline (DPBS)]
?. [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]
4 °C
?. [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 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 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 25 () 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 28 (), 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.