TITLE: Human Nasopharyngeal Swab Processing for Viable Single-Cell Suspension
AUTHORS: Ying Tang, Carly G.K. Ziegler, Vincent N. Miao, Andrew W. Navia, Joshua D. Bromley, Kenneth J. Wilson, Yilianys Pride, Mohammad Hasan, Taylor Christian, Hannah Laird, Anna Owings, Meredith Sloan, Haley B. Williams, Tanya O. Robinson, George E. Abraham III, Michal Senitko, Sarah C. Glover, Bruce Horwitz, Alex K. Shalek, Jose Ordovas-Montanes

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A protocol for recovering viable single cell suspensions from cryopreserved human nasopharyngeal swabs for downstream applications, such as single-cell RNA-seq. The illustrated schematic below details the process.</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/827a38476558c180e8f2b20f00d29c6343ad50cab638a3a10b74a6a571aeb656/cmjvbggt7.png" /></div></div>

[STEPS]
?. [Tube A]
Rapidly thaw cryovial (Tube A) in hands or thermal block set to .
37 °C
Carefully perform in accordance with your institute's safety guidelines. If handling potentially infectious material, inspect for cracks or leaks during warming
?. [Tube A]
Remove swab from Tube A using clean forceps, trim swab handle using scissors if necessary.
?. [Tube A]
Place swab in Tube B (15 mL conical), dip briefly to rinse swab.
?. [Tube A]
Move swab from Tube B (15 mL conical) to Tube C. Proceed directly to step 23 for Tube C.
?. [Tube A]
Transfer liquid in Tube A to Tube B (15 mL conical).
?. [Tube A]
Using  from Tube B (15 mL conical), wash Tube A.
[RPMI]
?. [Tube A]
Discard Tube A.
?. [Tube B]
Centrifuge Tube B (15 mL): .
Centrifuge: 400 34, 5 min, 4 10
?. [Tube B]
Remove supernatant with serological pipette.
?. [Tube B]
Resuspend pellet in .
[RPMI/10mM DTT]
?. [Tube B]
Transfer suspended cells from Tube B (15 mL) to Tube B (1.5 mL). Discard empty 15 mL conical.
?. [Tube B]
Place Tube B (1.5 mL) on thermomixer (37ºC, 300 rpm).
?. [Tube B]
Incubate for .
?. [Tube B]
Centrifuge Tube B (1.5 mL): .
Centrifuge: 400 34, 5 min, 4 10
?. [Tube B]
Remove supernatant with P1000 pipette.
?. [Tube B]
Resuspend pellet in .
[Accutase]
?. [Tube B]
Place Tube B (1.5 mL) on thermomixer (37ºC, 300 rpm).
?. [Tube B]
Incubate for .
?. [Tube C]
Place Tube C on thermomixer (37ºC, 300 rpm).
?. [Tube C]
Incubate for .
?. [Tube C]
Place swab in Tube D. Proceed directly to step 31 for Tube D.
?. [Tube C]
Centrifuge remaining liquid at .
Centrifuge: 400 34, 5 min, 4 10
?. [Tube C]
Remove supernatant with P1000 pipette.
?. [Tube C]
Resuspend pellet in .
[Accutase]
?. [Tube C]
Place Tube C on thermomixer (37ºC, 300 rpm).
?. [Tube C]
Incubate for .
?. [Tube D]
Place Tube D on thermomixer (37ºC, 300 rpm).
?. [Tube D]
Incubate for .
?. [After Tube B, C, and D's 30 minute Incubations]
After Tube B, Tube C, and Tube D have each finished their 30 minute incubations:
In practice, we wait until all tubes have finished their 30 minute incubation in Accutase to synchronize. We leave tubes on incubation for a maximum 50 minutes.
?. [Tube A]
Collect washing from Tube A in Tube B (15 mL conical).
?. [After Tube B, C, and D's 30 minute Incubations]
Place 70 µm cell strainer in a 50 mL conical.
?. [After Tube B, C, and D's 30 minute Incubations]
Wet cell strainer with .
[quenching buffer (RPMI/10% FBS/4 mM EDTA)]
?. [After Tube B, C, and D's 30 minute Incubations]
Pipette contents of Tube B, Tube C, and Tube D onto cell strainer.
Do not discard tubes.
?. [After Tube B, C, and D's 30 minute Incubations]
Use  to wash each Tube B, Tube C, and Tube D.
[quenching buffer]
?. [After Tube B, C, and D's 30 minute Incubations]
Add quenching buffer from Tube washes to cell strainer. Discard Tubes B, C, and D.
?. [After Tube B, C, and D's 30 minute Incubations]
Wash cell strainer with additional .
[quenching buffer]
?. [After Tube B, C, and D's 30 minute Incubations]
Discard cell strainer, cap 50 mL conical.
?. [After Tube B, C, and D's 30 minute Incubations]
Centrifuge 50 mL conical .
Centrifuge: 400 34, 10 min, 4 10
?. [After Tube B, C, and D's 30 minute Incubations]
Remove supernatant with serological pipette.
?. [After Tube B, C, and D's 30 minute Incubations]
Resuspend cell pellet in residual volume (often ~500 µL).
?. [After Tube B, C, and D's 30 minute Incubations]
Wash 50 mL conical with .
[RPMI/10% FBS]
?. [After Tube B, C, and D's 30 minute Incubations]
Centrifuge Tube E .
Centrifuge: 400 34, 5 min, 4 10
?. [After Tube B, C, and D's 30 minute Incubations]
Remove supernatant with P1000 pipette.
?. [After Tube B, C, and D's 30 minute Incubations]
Resuspend pellet in .
[RPMI/10% FBS]
?. [After Tube B, C, and D's 30 minute Incubations]
Manually agitate the swab in Tube D in the quenching buffer to ensure full rinse.
?. [After Tube B, C, and D's 30 minute Incubations]
Transfer washing from 50 mL conical to Tube E.
?. [Count cells from Tube E]
In 96 well plate, add .
[trypan blue]
?. [Count cells from Tube E]
Add  to well containing trypan blue.
[cells from Tube E]
?. [Count cells from Tube E]
Pipette to mix cells in trypan blue, transfer  to hemocytometer port.
10 µl
?. [Count cells from Tube E]
Count viable cells across 4 quadrants.
?. [Count cells from Tube E]
Record total cell number and calculate cell concentration.
?. [Count cells from Tube E]
Take photo of cells at 20x.
?. [Count cells from Tube E]
Load 20,000 viable cells on Seq-Well array.
Or load total volume of Tube E if total cell count is lower than 20,000.
?. [Population Lysates]
Label cryovials and add  to each tube.
[RLT/1% BME]
?. [Population Lysates]
Add 20,000 viable cells to each tube. Tap or pipette to mix cell suspension with RLT/1% BME
?. [Population Lysates]
Snap freeze on dry ice for at least .
?. [Population Lysates]
Transfer to .
-80 °C
?. [Before You Start]
Prepare and label a 15 mL conical as Tube B with .
[RPMI]
?. [Before You Start]
Prepare and label a 1.5 mL tube as Tube C with .
[ RPMI/10 mM DTT]
?. [Before You Start]
Prepare and label a 1.5 mL tube as Tube D with .
[Accutase]
?. [Before You Start]
Prepare and label a 1.5 mL tube also as Tube B, leave this one empty.
?. [After Tube B, C, and D's 30 minute Incubations]
Transfer resuspended cells from 50 mL conical to Tube E (1.5 mL tube).