Oct 31, 2020
Human Nasopharyngeal Swab Processing for Viable Single-Cell Suspension
- Ying Tang1,
- Carly G.K. Ziegler2,
- Vincent N. Miao2,
- Andrew W. Navia2,
- Joshua D. Bromley2,
- Kenneth J. Wilson3,
- Yilianys Pride3,
- Mohammad Hasan3,
- Taylor Christian3,
- Hannah Laird3,
- Anna Owings3,
- Meredith Sloan3,
- Haley B. Williams3,
- Tanya O. Robinson3,
- George E. Abraham III3,
- Michal Senitko3,
- Sarah C. Glover3,
- Bruce Horwitz1,
- Alex K. Shalek2,
- Jose Ordovas-Montanes1
- 1Boston Children's Hospital;
- 2Massachusetts Institute of Technology;
- 3University of Mississippi Medical Center
- Human Cell Atlas Method Development Community
- Coronavirus Method Development Community
Protocol Citation: 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 2020. Human Nasopharyngeal Swab Processing for Viable Single-Cell Suspension. protocols.io https://dx.doi.org/10.17504/protocols.io.bjhkkj4w
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
Created: August 09, 2020
Last Modified: October 31, 2020
Protocol Integer ID: 40204
Keywords: Nasal Swab, COVID-19, Nasopharyngeal Swab, single-cell RNA-seq, nasal epithelia, nasopharynx, sars-cov-2, cryopreserved human nasopharyngeal swab, recovering viable single cell suspension, human nasopharyngeal swab processing, cell suspension, cell rna, rna
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Abstract
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.
Attachments
Image Attribution
Created with BioRender
Guidelines
Samples should be collected by a trained medical professional using a nasal swab (FLOQSwabs, Copan flocked swabs) in accordance with the manufacturer’s instructions. Briefly, the process was performed as follows. First, the patient’s head was tilted back slightly, and the swab was inserted along the nasal septum, above the floor of the nasal passage to the nasopharynx until slight resistance was felt. The swab was then left in place for several seconds to absorb secretions and slowly removed while rotating. The swab was placed in a 1.7 mL cryovial containing 90% fetal bovine serum (FBS)/10% dimethyl sulfoxide (DMSO) and frozen using a slow-cooling device (Thermo Scientific Mr. Frosty Freezing Container) at -80 °C, and stored in liquid nitrogen.
Materials
For processing of 1 cryopreserved nasopharyngeal swab:
- 2.5 mL of RPMI/10 mM Dithiothreitol (DTT) (made fresh)
- 3.5 mL of Accutase
- 6 mL RPMI
- 8 mL quenching media containing RPMI/10% fetal bovine serum (FBS)/4 mM EDTA
- 2 mL RPMI/10% FBS
- 15 mL conical labeled Tube B containing 5 mL RPMI
- 1.5 mL tube labeled Tube B, empty
- 1.5 mL tube labeled Tube C, with 1 mL RPMI/10 mM DTT
- 1.5 mL tube labeled Tube D, with 1 mL Accutase
- 50 mL conical
- 70 µm cell strainer that fits 50 mL conical
- 1.5 mL tube labeled Tube E
- Forceps and scissors
- Thermomixer set to 37ºC, agitating at 300 rpm
- 10 µL trypan blue
- NI hemocytometer
- 96 well plate for cell counting
- RLT buffer (Qiagen)/1% 2-mercaptoethanol (BME)
- Cryovials or snap-top ependorf tubes for population lysates
Troubleshooting
Safety warnings
For hazard information and safety warnings regarding nasopharyngeal swabs or any listed materials, please refer to the SDS (Safety Data Sheet).
For samples obtained from individuals diagnosed with, or at any risk of, an infection (e.g. SARS-CoV-2), additional precautions should be taken in accordance with your institute's regulations on biosafety. These include elimination of aerosol generating steps where possible (e.g., no vacuum aspiration), all steps prior to cell lysis should be carried out in a biosafety cabinet, including thermomixing and centrifugation where possible. When necessary, samples should only be removed from the biosafety cabinet in decontaminated and sealed secondary containment. Personal protective equipment including a gown, two pairs of non-sterile gloves, a protective surgical or N95 mask, and a face shield should be worn during sample processing.
Before You Start
Prepare and label a 15 mL conical as Tube B with 5 mL RPMI .
Prepare and label a 1.5 mL tube also as Tube B, leave this one empty.
Prepare and label a 1.5 mL tube as Tube C with 1 mL RPMI/10 mM DTT .
Prepare and label a 1.5 mL tube as Tube D with 1 mL Accutase .
Tube A
Rapidly thaw cryovial (Tube A) in hands or thermal block set to 37 °C .
Safety information
Carefully perform in accordance with your institute's safety guidelines. If handling potentially infectious material, inspect for cracks or leaks during warming
Remove swab from Tube A using clean forceps, trim swab handle using scissors if necessary.
Place swab in Tube B (15 mL conical), dip briefly to rinse swab.
Move swab from Tube B (15 mL conical) to Tube C. Proceed directly to step 23 for Tube C.
Transfer liquid in Tube A to Tube B (15 mL conical).
Using 1 mL RPMI from Tube B (15 mL conical), wash Tube A.
Collect washing from Tube A in Tube B (15 mL conical).
Discard Tube A.
Tube B
Centrifuge Tube B (15 mL): 400 x g, 4°C, 00:05:00 .
Remove supernatant with serological pipette.
Resuspend pellet in 1 mL RPMI/10mM DTT .
Transfer suspended cells from Tube B (15 mL) to Tube B (1.5 mL). Discard empty 15 mL conical.
Place Tube B (1.5 mL) on thermomixer (37ºC, 300 rpm).
Incubate for 00:15:00 .
Centrifuge Tube B (1.5 mL): 400 x g, 4°C, 00:05:00 .
Remove supernatant with P1000 pipette.
Resuspend pellet in 1 mL Accutase .
Place Tube B (1.5 mL) on thermomixer (37ºC, 300 rpm).
Incubate for 00:30:00 .
Tube C
Place Tube C on thermomixer (37ºC, 300 rpm).
Incubate for 00:15:00 .
Place swab in Tube D. Proceed directly to step 31 for Tube D.
Centrifuge remaining liquid at 400 x g, 4°C, 00:05:00..
Remove supernatant with P1000 pipette.
Resuspend pellet in 1 mL Accutase .
Place Tube C on thermomixer (37ºC, 300 rpm).
Incubate for 00:30:00 .
Tube D
Place Tube D on thermomixer (37ºC, 300 rpm).
Incubate for 00:30:00 .
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:
Note
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.
Place 70 µm cell strainer in a 50 mL conical.
Wet cell strainer with 3 mL quenching buffer (RPMI/10% FBS/4 mM EDTA) .
Pipette contents of Tube B, Tube C, and Tube D onto cell strainer.
Note
Do not discard tubes.
Use 1 mL quenching buffer to wash each Tube B, Tube C, and Tube D.
Manually agitate the swab in Tube D in the quenching buffer to ensure full rinse.
Add quenching buffer from Tube washes to cell strainer. Discard Tubes B, C, and D.
Wash cell strainer with additional 2 mL quenching buffer .
Discard cell strainer, cap 50 mL conical.
Centrifuge 50 mL conical 400 x g, 4°C, 00:10:00 .
Remove supernatant with serological pipette.
Resuspend cell pellet in residual volume (often ~500 µL).
Transfer resuspended cells from 50 mL conical to Tube E (1.5 mL tube).
Wash 50 mL conical with 500 µL RPMI/10% FBS .
Transfer washing from 50 mL conical to Tube E.
Centrifuge Tube E 400 x g, 4°C, 00:05:00 .
Remove supernatant with P1000 pipette.
Resuspend pellet in 200 µL RPMI/10% FBS .
Count cells from Tube E
In 96 well plate, add 10 µL trypan blue .
Add 10 µL cells from Tube E to well containing trypan blue.
Pipette to mix cells in trypan blue, transfer 10 µL to hemocytometer port.
Count viable cells across 4 quadrants.
Record total cell number and calculate cell concentration.
Take photo of cells at 20x.
Load 20,000 viable cells on Seq-Well array.
Note
Or load total volume of Tube E if total cell count is lower than 20,000.
Population Lysates
Label cryovials and add 100 µL RLT/1% BME to each tube.
Add 20,000 viable cells to each tube. Tap or pipette to mix cell suspension with RLT/1% BME
Snap freeze on dry ice for at least 00:10:00 .
Transfer to -80 °C .