Dec 22, 2022
CZI Pediatric Nasopharyngeal Swab Processing for 10X scRNA-seq (Illustrated Protocol)
- Jaclyn M Long1,
- Erica M Langan1,
- 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
Protocol Citation: Jaclyn M Long, Erica M Langan, 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 2022. CZI Pediatric Nasopharyngeal Swab Processing for 10X scRNA-seq (Illustrated Protocol). protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr49jogmk/v1
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: December 19, 2022
Last Modified: December 22, 2022
Protocol Integer ID: 74234
Keywords: Nasal Swab, COVID-19, Nasopharyngeal Swab, single-cell RNA-seq, nasal epithelia, nasopharynx, sars-cov-2
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Abstract
A protocol for recovering viable single cell suspensions from cryopreserved human nasopharyngeal swabs for pooled single-cell RNA-seq and individual bulk 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
Materials & Reagents for 1 swab (with overages):
- 2.5 mL of RPMI + 10 mM Dithiothreitol (DTT) (made fresh)
- 3.5 mL of Accutase
- 6 mL RPMI
- 8 mL quenching buffer containing RPMI + 10% fetal bovine serum (FBS) + 4 mM EDTA
- 2 mL RPMI + 10% FBS
- 15 mL conical labeled Tube B containing 2 mL RPMI (previously 5 mL)
- 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
- 1.5 mL tube for counting
- 1.5 mL tube for pooling cells (if doing 3 samples, need 1 tube)
- 10 µL trypan blue
- 1 mL PBS.+ 1% BSA
- 15 mL conical for collecting viral supernatant labeled Tube F
- 3 cryovials for viral supernatant lysate
- 3 96-well PCR plates or 3 cryovials for bulk RNAseq lysate
- RLT + 1% 2-mercaptoethanol (BME)
Equipment
- Forceps and scissors
- Thermomixer set to 37ºC, agitating at 300 rpm
- Hemocytometer slides
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
Before You Start
Record sample characteristics in Table 1
| Site ID | Study ID | Case/Control | Virus | Date of Enrollment | Sex | Age | Sample ID | Batch | Pool | |
Prepare 50 mL conicals with necessary reagents:
- 1X RPMI + 10 mM DTT
- 1X Accutase
- RPMI + 10% FBS + 4 mM EDTA (quenching buffer)
- RPMI + 10% FBS
Label 2 sets of 15 mL conicals (Tube B & Tube F). Prepare Tube B with 2 mL RPMI .
Tube B and Tube F setup for 6 swabs
Label 4 sets of 1.5 mL tubes (Tube B, Tube C, Tube D, Tube E). Prepare Tube C with 1 mL RPMI + 10 mM DTT and Tube D with 1 mL Accutase
Label 1 set of 50 mL tubes for filtering at later step (can set aside for beginning of protocol)
Final Set-Up
Final Set-Up
Timing
Timing
Fill out timing chart at indicated steps of protocol
| Step | Time | |
| Start time: | ||
| Time after preparation of single cell suspension: | ||
| Time after counting: | ||
| Time 10X controller run started: | ||
| Time 2nd 10X controller run started (if applicable): | ||
| Time lysis buffer added for bulk lysates: |
Tube A (the cryovial)
Tube A (the cryovial)
Rapidly (within 1-2 minutes) thaw cryovial (Tube A) in 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.
Step 9-11 - first swab transfer.mp4 Dip swab in Tube B (15 mL conical) once. Swirl briefly (approximately 3 seconds) to rinse swab.
Move swab from Tube B (15 mL conical) to Tube C. Trim swab handle using scissors if necessary so that it can fit inside a 1.5mL tube (see video). Proceed directly to step 27 for Tube C. In parallel, continue processing Tube B.
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). Continue below with Tube B
Discard Tube A.
Tube B
Tube B
Centrifuge Tube B (15 mL): 400 x g, 4°C, 00:05:00 .
Remove supernatant with serological pipette or P1000. Do not discard supernatant.
Transfer supernatant to Tube F.
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 . In parallel, continue processing Tube C.
Centrifuge Tube B (1.5 mL): 400 x g, 4°C, 00:05:00 .
Remove supernatant with P1000 pipette. Discard supernatant.
Resuspend pellet in 1 mL Accutase .
Place Tube B (1.5 mL) on thermomixer (37ºC, 300 rpm).
Incubate for 00:30:00 .
Tube C
Tube C
Place Tube C on thermomixer (37ºC, 300 rpm).
Incubate for 00:15:00 .
Place swab in Tube D. Proceed directly to step 36 for Tube D.
Step 29 - second swab transfer.mp4 Centrifuge remaining liquid at 400 x g, 4°C, 00:05:00..
Remove supernatant with P1000 pipette. Do not discard supernatant.
Place supernatant in Tube F. Keep Tube F on ice until you proceed to Step 71.
Resuspend pellet in 1 mL Accutase .
Place Tube C on thermomixer (37ºC, 300 rpm).
Incubate for 00:30:00 .
Tube D
Tube D
Place Tube D on thermomixer (37ºC, 300 rpm).
Incubate for 00:30:00 .
Note
Take 10X reagents out at this step so that they have time to equilibrate to the appropriate temperature.
After Tube B, C, and D's 30 minute Incubations
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 filter in 50 mL conical.
Wet filter with 3 mL quenching buffer (RPMI + 10% FBS + 4 mM EDTA) .
Pipette contents of Tube B, Tube C, and Tube D onto filter with P1000 pipette
Step 41 - adding Tubes B,C,D contents to filter.mp4 Note
Do not discard original 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 media with forceps to ensure full rinse.
Add quenching buffer from washes to filter. Discard Tubes B, C, and D.
Wash filter with additional 2 mL quenching buffer .
Discard filter, cap 50 mL conical.
Centrifuge 50 mL conical 400 x g, 4°C, 00:10:00 .
Note
It will likely be very challenging to see a pellet in the 50 mL conical at this point
Remove supernatant with serological pipette. Leave ~500 µL in the bottom of the tube.
Approximate residual volume to aim for
Note
Remove supernatant carefully at this step! You don't want to pipette up your cells but you will need to add the volume you leave in this 50 mL conical and 1 mL RPMI + 10% FBS to a 1.5 mL tube in the next steps, so try not to exceed 500 µL residual volume.
Add 500 µL RPMI + 10% FBS to the tube to resuspend cells.
Transfer resuspended cells (~ 1 mL) 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 1 mL RPMI + 10% FBS .
Example pellet in Tube E after centrifuging
Note
At this point, you should be able to see a reasonably-sized pellet!
Centrifuge Tube E 400 x g, 4°C, 00:05:00
5m
Resuspend pellet in 200 µL RPMI + 10% FBS
Count cells from Tube E
Count cells from Tube E
In a new empty 1.5 mL tube, add 10 µL trypan blue .
Add 10 µL cells from Tube E to 1.5 mL tube containing trypan blue.
Pipette to mix cells in trypan blue, transfer 10 µL to hemocytometer port.
Count viable cells across 8 quadrants. One quadrant is the corner of a hemocytometer grid, consisting of 16 smaller squares.
Record total cell number and calculate cell concentration using the "Cell Counts & 10X Volumes" sheet of the Calculation Tables template.
Calculation Tables template.xlsx Take photo of cells at 4X. This can be done using a camera attached to the microscope or with a phone placed adjacent to the microscope lens.
Representative hemocytometer image at 4X
Prepare cells for 10X
Prepare cells for 10X
Prepare 1.5 mL tube for each pool
For each sample, add "Volume Necessary for Desired Number of Cells" from "Cell Counts & 10X Volumes" sheet of the Calculation Tables template to correct pool.
Pipette cells to mix
If volume of pool is > 100 µL, centrifuge cells 400 x g, 4°C, 00:05:00 , then resuspend in 500 µL PBS + 1% BSA and proceed to next step.
If volume of pool is < 100 µL, add 500 µL PBS + 1% BSA directly to pool and proceed to next step.
5m
Centrifuge cells 400 x g, 4°C, 00:05:00
5m
Resuspend cells in 43.3 µL PBS + 1% BSA (Total volume of cell suspension + water on page 27 of 10X Protocol).
Add 43.3 µL cell suspension directly to 31.9 µL master mix (Step 1.2b of 10X Protocol)
Proceed with instructions in 10X Protocol to load the chip and run the controller (through Step 1.3)
At Step 1.4f, take a picture of the GEMs in the pipette tips.
Example photo of successful run
Tube F Processing (Viral Lysates)
Tube F Processing (Viral Lysates)
Remove a 200uL aliquot from Tube F and transfer to a new, empty 1.5mL tube. Store at -80C for antibody studies.
Note
In Tube F, you should have 2 mL from original RPMI + 0.5-1 mL from swab cryopreservative + 1 mL from Tube C supernatant = ~4 mL total
Add 1 mL RLT + 1% BME to Tube F.
Distribute contents of Tube F into 3 cryovials per sample
Note
You can save time here by labeling cryovials/plates for viral and bulk lysates the day before!
Snap freeze on dry ice
Store at -80 °C
Lysates for Bulk RNA-seq
Lysates for Bulk RNA-seq
40m
40m
For each lysate, add "Volume Necessary for Each Lysate" from "Bulk RNA Lysate Volumes" sheet of the Calculation Tables template to one well of a 96-well PCR plate according to the Lysate Storage Plate map.
Note
Typically, we aim to store 3 lysates per sample. To do so, we label 3 plates and fill 1 well per sample in each plate, if cell numbers allow. Make sure to record which well has which sample.
Seal plates and centrifuge 400 x g, 4°C, 00:05:00
5m
Aspirate media
Note
It may be difficult to see a pellet here, so it's okay to leave some residual volume before resuspending in lysis buffer in the next step
Resuspend cells in 50 µL RLT + 1% BME . Mix with pipette. Bubbles are okay here.
Seal plate with foil seal and spin down briefly.
Place plate on dry ice for 00:15:00 -00:20:00 to snap-freeze lysate.
35m
Store at -80 °C until ready to use.