TITLE: CZI Pediatric Nasopharyngeal Swab Processing for 10X scRNA-seq (Illustrated Protocol)
AUTHORS: 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

[DESCRIPTION]
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.
 
  


[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.

[STEPS]
SECTION: Tube A (the cryovial)
8. Rapidly (within 1-2 minutes) thaw cryovial (Tube A) in thermal block set to 37 °C.
 

SECTION: Tube A (the cryovial)
9. Remove swab from Tube A using clean forceps.
  

SECTION: Tube A (the cryovial)
10. Dip swab in Tube B (15 mL conical) once. Swirl briefly (approximately 3 seconds) to rinse swab.
SECTION: Tube A (the cryovial)
11. 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.


SECTION: Tube A (the cryovial)
12. Transfer liquid in Tube A to Tube B (15 mL conical).
SECTION: Tube A (the cryovial)
13. Using 1 mL from Tube B (15 mL conical), wash Tube A. 

SECTION: Tube A (the cryovial)
14. Discard Tube A.
SECTION: Tube B
15. Centrifuge Tube B (15 mL): 400 x g, 5 min, 4 °C.
SECTION: Tube B
16. Remove supernatant with serological pipette or P1000. Do not discard supernatant.

SECTION: Tube B
18. Resuspend pellet in 1 mL.

SECTION: Tube B
19. Transfer suspended cells from Tube B (15 mL) to Tube B (1.5 mL). Discard empty 15 mL conical.
SECTION: Tube B
20. Place Tube B (1.5 mL) on thermomixer (37ºC, 300 rpm).
SECTION: Tube B
21. Incubate for 15 min. In parallel, continue processing Tube C.

SECTION: Tube B
22. Centrifuge Tube B (1.5 mL): 400 x g, 5 min, 4 °C.

SECTION: Tube B
23. Remove supernatant with P1000 pipette. Discard supernatant.
SECTION: Tube B
24. Resuspend pellet in 1 mL.

SECTION: Tube B
25. Place Tube B (1.5 mL) on thermomixer (37ºC, 300 rpm).
SECTION: Tube B
26. Incubate for 30 min.

SECTION: Tube C
27. Place Tube C on thermomixer (37ºC, 300 rpm).
SECTION: Tube C
28. Incubate for 15 min.

SECTION: Tube C
29. Place swab in Tube D. Proceed directly to step 36 for Tube D.
  

SECTION: Tube C
30. Centrifuge remaining liquid at 400 x g, 5 min, 4 °C.

SECTION: Tube C
31. Remove supernatant with P1000 pipette. Do not discard supernatant.
SECTION: Tube C
33. Resuspend pellet in 1 mL.

SECTION: Tube C
34. Place Tube C on thermomixer (37ºC, 300 rpm).
SECTION: Tube C
35. Incubate for 30 min.

SECTION: Tube D
36. Place Tube D on thermomixer (37ºC, 300 rpm).
SECTION: Tube D
37. Incubate for 30 min.

 

SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
38. After Tube B, Tube C, and Tube D have each finished their 30 minute incubations:
 

SECTION: Tube A (the cryovial)
13.1. Collect washing from Tube A in Tube B (15 mL conical). Continue below with Tube B
SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
39. Place 70 µm filter in 50 mL conical.
SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
40. Wet filter with 3 mL.

SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
41. Pipette contents of Tube B, Tube C, and Tube D onto filter with P1000 pipette
  
 


SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
42. Use 1 mL to wash each Tube B, Tube C, and Tube D. 

SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
43. Add quenching buffer from washes to filter. Discard Tubes B, C, and D.
SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
44. Wash filter with additional 2 mL.

SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
45. Discard filter, cap 50 mL conical.
SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
46. Centrifuge 50 mL conical 400 x g, 10 min, 4 °C.

    
 


SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
47. Remove supernatant with serological pipette. Leave ~500 µL in the bottom of the tube.
  

 

SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
48. Add 500 µL RPMI + 10% FBS to the tube to resuspend cells.
SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
50. Wash 50 mL conical with 500 µL. 
SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
51. Centrifuge Tube E 400 x g, 5 min, 4 °C.

SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
52. Remove supernatant with P1000 pipette.
SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
53. Resuspend pellet in 1 mL.
  

 

SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
42.1. Manually agitate the swab in Tube D in the quenching media with forceps to ensure full rinse.
SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
50.1. Transfer washing from 50 mL conical to Tube E.

SECTION: Count cells from Tube E
56. In a new empty 1.5 mL tube, add 10 µL.

SECTION: Count cells from Tube E
57. Add 10 µL to 1.5 mL tube containing trypan blue.

SECTION: Count cells from Tube E
58. Pipette to mix cells in trypan blue, transfer 10 µL to hemocytometer port.

SECTION: Count cells from Tube E
59. Count viable cells across 8 quadrants. One quadrant is the corner of a hemocytometer grid, consisting of 16 smaller squares. 
SECTION: Count cells from Tube E
60. Record total cell number and calculate cell concentration using the "Cell Counts & 10X Volumes" sheet of the Calculation Tables template.
   

SECTION: Lysates for Bulk RNA-seq
76. 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. 
 

SECTION: Before You Start
3. Label 2 sets of 15 mL conicals (Tube B & Tube F). Prepare Tube B with 2 mL.
  

SECTION: Before You Start
4. Label 4 sets of 1.5 mL tubes (Tube B, Tube C, Tube D, Tube E). Prepare Tube C with 1 mL and Tube D with 1 mL 
   

SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
49. Transfer resuspended cells (~ 1 mL) from 50 mL conical to Tube E (1.5 mL tube).
SECTION: Before You Start
1. Record sample characteristics in Table 1

 

SECTION: Before You Start
2. 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

  


5. Label 1 set of 50 mL tubes for filtering at later step (can set aside for beginning of protocol)
  

SECTION: Final Set-Up
6.   

SECTION: Timing
7. Fill out timing chart at indicated steps of protocol

 

SECTION: Tube C
32. Place supernatant in Tube F.  Keep Tube F on ice until you proceed to Step 71.
SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
54. Centrifuge Tube E 400 x g, 5 min, 4 °C 

SECTION: After Tube B, C, and D&#x27;s 30 minute Incubations
55. Resuspend pellet in 200 µL RPMI + 10% FBS
SECTION: Count cells from Tube E
61. 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. 
      


SECTION: Prepare cells for 10X
62. Prepare 1.5 mL tube for each pool
SECTION: Prepare cells for 10X
63. 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.
SECTION: Prepare cells for 10X
64. Pipette cells to mix


66. 
SECTION: Prepare cells for 10X
67. 
SECTION: Lysates for Bulk RNA-seq
77. Seal plates and centrifuge 400 x g, 5 min, 4 °C 

SECTION: Lysates for Bulk RNA-seq
78. Aspirate media

 

SECTION: Prepare cells for 10X
65. If volume of pool is > 100  µL, centrifuge cells 400 x g, 5 min, 4 °C , then resuspend in 500 µL and proceed to next step.

If volume of pool is < 100  µL, add 500 µL directly to pool and proceed to next step.

SECTION: Prepare cells for 10X
68. 
SECTION: Prepare cells for 10X
65. 
SECTION: Prepare cells for 10X
66. Centrifuge cells 400 x g, 5 min, 4 °C 

SECTION: Prepare cells for 10X
67. Resuspend cells in 43.3 µL  (Total volume of cell suspension + water on page 27 of 10X Protocol).

SECTION: Prepare cells for 10X
68. Add 43.3 µL directly to 31.9 µL (Step 1.2b of 10X Protocol) 

SECTION: Prepare cells for 10X
69. Proceed with instructions in 10X Protocol to load the chip and run the controller (through Step 1.3)
SECTION: Prepare cells for 10X
70. At Step 1.4f, take a picture of the GEMs in the pipette tips.
  

SECTION: Tube F Processing (Viral Lysates)
72. 
 
Add 1 mL to Tube F.

SECTION: Tube F Processing (Viral Lysates)
73. Distribute contents of Tube F into 3 cryovials per sample
 

SECTION: Tube F Processing (Viral Lysates)
74. Snap freeze on dry ice
SECTION: Tube F Processing (Viral Lysates)
75. Store at -80 °C 

SECTION: Lysates for Bulk RNA-seq
79. Resuspend cells in 50 µL . Mix with pipette. Bubbles are okay here.

SECTION: Lysates for Bulk RNA-seq
80. Seal plate with foil seal and spin down briefly.
SECTION: Lysates for Bulk RNA-seq
81. Place plate on dry ice for 15 min -20 min  to snap-freeze lysate.

SECTION: Lysates for Bulk RNA-seq
82. Store at -80 °C until ready to use.

SECTION: Tube B
17. Transfer supernatant to Tube F.
SECTION: Tube F Processing (Viral Lysates)
71. Remove a 200uL aliquot from Tube F and transfer to a new, empty 1.5mL tube. Store at -80C for antibody studies.