TITLE: SARS-CoV-2 inactivation and scRNAseq sample preparation protocol
AUTHORS: Raven M Osborn, Justin Leach, Michelle Zanche, John M. Ashton, ChinYi Chu, Juilee Thakar, Stephen Dewhurst, Sonia Rosenberger, Martin Pavelka, Gloria S Pryhuber, Thomas J. Mariani, Christopher Anderson

[DESCRIPTION]
Coronavirus disease (COVID-19) is an infectious disease caused by the SARS coronavirus 2 (SARS-CoV-2) virus. Direct assessment, detection, and quantitative analysis using high throughput methods like single-cell RNA sequencing (scRNAseq) is imperative to understanding the host response to SARS-CoV-2. One barrier to studying SARS-CoV-2 in the laboratory setting is the requirement to process virus-infected cell cultures, and potentially infectious materials derived therefrom, under Biosafety Level 3 (BSL-3) containment. However, BSL3 laboratory facilities are rare and only a subset of these facilities are outfitted with the equipment needed to perform high-throughput molecular assays. Here, we describe a method for preparing non-hazardous RNA samples from SARS-CoV-2 infected cells, that enables scRNAseq analyses to be conducted safely in a BSL2 facility – thereby making molecular assays of SARS-CoV-2 cells accessible to a much larger community of researchers.

[GUIDELINES]
Guidelines:

This procedure must be performed in a biosafety cabinet according to the approval and Standard Operation Procedures for your institution’s Biosafety Level 3 program.

This protocol was created in the University of Rochester’s Center for Advanced Research Technologies Biosafety Level 3 (BSL3) facility, under the University of Rochester’s Institutional Biosafety Committee (IBC) oversight.

Notes:
This procedure assumes a SARS-CoV-2 – infected cell culture as a starting point. 
No viable virus can be taken out of the BSL3 facility unless inactivated.
All pipet tips are filter barrier tips. Follow procedures in “Pipetting”. 
No vacuum suction is used to remove media or wash buffers from tubes or tissue culture wells; only use pipettors.
Troughs for multichannel pipettors are only used with media, not virus suspensions. Tips from multichannel pipets are rinsed in a container of disinfectant and then ejected into a biohazard bag in a container. Both containers must be wide enough to fit the pipettor.
No work is conducted on an open bench, and all cultures are in secondary containers when outside of the biosafety cabinet. The only exception is when culture trays or flasks are examined with the inverted microscope. In these instances, use of a Powered Air Purifying Respirator (PAPR) is required. 
All secondary containers should be labeled with your name, date, and “SARS-CoV-2”. 


[STEPS]
SECTION: Lifting cells from tissue culture plate
1. Set up the biosafety cabinet according to your institution’s BSL3 biosafety cabinet setup standard operating procedure or the "Basic Biosafety Cabinet Setup” supplied in this protocol. 
 

SECTION: Lifting cells from tissue culture plate
2. Transfer the tissue culture plates, in a secondary container, from the CO2 incubator to the biosafety cabinet.
SECTION: Lifting cells from tissue culture plate
3. If supernatant from infected cells is of interest, collect supernatant into screw-capped microcentrifuge tubes and incubate at 65 °C for 60 min to inactivate the virus.

SECTION: Lifting cells from tissue culture plate
4. Wash cells by dispensing and aspirating HEPES buffered saline solution (Lonza CC-5022).
SECTION: Lifting cells from tissue culture plate
5. Add 500 µL Trypsin-EDTA (Lonza Catalog #: CC-5012) and incubate for 10 min at 37 °C.

SECTION: Lifting cells from tissue culture plate
6. Gently pipette up and down to dissociate cells using wide-bore 200-ul pipette tip.
SECTION: Lifting cells from tissue culture plate
7. Transfer cells to a 1.5-ml tube containing 500 µL of ice-cold Trypsin-Neutralization-Solution (CC-5002).

SECTION: Lifting cells from tissue culture plate
8. If needed, add an additional 500 µL Trypsin-EDTA to cells and incubate at 37 °C for no longer than 30 min to maximize collection of cells.

SECTION: Lifting cells from tissue culture plate
9. Transfer cells to the same cold Trypsin-Neutralization-Solution 1.5-ml tube.
SECTION: Lifting cells from tissue culture plate
10. Centrifuge cells at 300 x g, 5 min according to your institution’s BSL3 centrifugation procedures or the “Centrifugation” procedure supplied in this protocol. Remove the supernatant without disrupting the cell pellet.

SECTION: Lifting cells from tissue culture plate
11. Using a wide-bore pipette tip, add 1 mL chilled HEPES and gently pipette mix 10x or until cells are resuspended.

SECTION: Lifting cells from tissue culture plate
12. Centrifuge cells at 300 x g, 5 min.

SECTION: SARS-CoV-2 inactivation with 1:1 methanol-acetone
13. 
 
Remove the supernatant without disrupting the cell pellet.
SECTION: SARS-CoV-2 inactivation with 1:1 methanol-acetone
14. Using a wide-bore pipette tip, add 100 µL chilled 1X DPBS and gently pipette mix 10x or until cells are resuspended.

SECTION: SARS-CoV-2 inactivation with 1:1 methanol-acetone
15. In a dropwise fashion, add chilled 1 mL 1:1 methanol acetone mixture to cells. 
 

SECTION: SARS-CoV-2 inactivation with 1:1 methanol-acetone
16. Incubate on ice for 60 min.

SECTION: Single cell RNAseq preparation
17. 
 
Centrifuge cells at 1000 x g, 5 min, 4 °C.

SECTION: Single cell RNAseq preparation
18. Remove the methanol-acetone mixture without disrupting the cell pellet.
SECTION: Single cell RNAseq preparation
19. Resuspend cells in 500 µL of ice-cold SSC cocktail (3× Lonza AccuGENE SSC-0.04% BSA + 1 millimolar (mM) DTT + 0.2 U/μL RNase Inhibitor).

SECTION: Single cell RNAseq preparation
20. Check that cells are fixed using trypan blue (90 µL of trypan blue with 10 µL of cells).

SECTION: Single cell RNAseq preparation
21. Count cells on a disposable c-chip hemocytometer contained in a petri dish, and resuspend cells to a final density of about 2000 cells/μl in the SSC cocktail.
SECTION: Single cell RNAseq preparation
22. Proceed to standard scRNAseq protocol.
SECTION: Additional Procedures: Standard item removal procedure
24. 
 
The following applies to the removal of items such as: metal cans, pipet boxes, any secondary container, sealed centrifuge buckets or rotors, and the electroporation chamber unit. 
SECTION: Additional Procedures: Standard item removal procedure
25. For vortexers and other small equipment:
SECTION: Additional Procedures: Standard item removal procedure
26. For tube racks: 

Racks are placed into a waste bag, which is folded over itself. The bag is removed from the biological safety cabinet with the standard item removal procedure into another bag and closed with a rubber band. 
The outer bag should be labeled “SAVE”. It is then ready for the autoclave. 
SECTION: Additional Procedures: Pipetting and other liquid transfers
27. Use mechanical pipetting devices only. NO MOUTH PIPETTING is permitted. 
Do not forcefully eject liquid from pipettes and avoid expelling the last drop from the pipette. 
When dispensing the liquid from a pipette, the tip should be below the fluid level or as close as possible to the agar level. Splatter from dropping liquid will create aerosols. 
Aerosol barrier tips should be used with pipetters. 
Re-suspension of viable material must be done so the tip is below the liquid level. 
The transfer of infectious liquids must be done in ways that reduce the generation of aerosols. 
Transfers using pipettes or pipetmen should always be done with the tip of the pipette or pipette tip positioned below the top of the liquid as it is flowing into the receptacle. Do not blow out the last drop. 
Transfer of cultures from culture vessels into tubes should always be done with pipettes and should never be poured. Washes may be poured off into a waste container in the biological safety cabinet. 
Aerosol barrier tips should be used with pipettes. 
No glass pipettes should be used, with the exception of 6-inch Pasteur pipettes, which are only used to remove liquid from electroporation cuvettes. Waste Pasteur pipettes are disposed of in a sharps container. 
After transfer with a pipette or pipette tip, pull up a quantity of disinfectant into pipette or tip and then flush out. Eject tip into waste container in the biological safety cabinet or place the pipette into the waste container in the biological safety cabinet.
SECTION: Additional Procedures: Centrifugation
28. 
 
The following procedures must be followed during centrifugation.
Only use appropriate tubes/bottles and check maximum centrifuging speed.
Before centrifuging, inspect tubes/bottles for cracks and stress marks. 
Make sure the correct adapters are in place. 
Fill and decant all centrifuge tubes/bottles within the biological safety cabinet. Wipe outside with disinfectant before placing into the rotor. 
If decontamination of a rotor or bucket is required, soak rotor / buckets and lids in a container of disinfectant for 10 min in the biological safety cabinet. Rinse with water. Then wipe down, replace cover, and remove from the biological safety cabinet with the standard procedures. 

SECTION: Additional Procedures: Tabletop Centrifugation - Swinging Bucket
29. Centrifugation in the tabletop centrifuge is always done with the aerosol proof buckets with gasketed caps. The sealed buckets are never opened outside of the biological safety cabinet, regardless of whether or not they contain tubes.
SECTION: Additional Procedures: Tabletop Centrifugation - Swinging Bucket
30. Take the buckets or rotors into the biological safety cabinet and open it.
SECTION: Additional Procedures: Tabletop Centrifugation - Swinging Bucket
31. After centrifugation, take the buckets to the biological safety cabinet and remove the lids. Remove the tubes from the buckets and wipe down the inside of the lids with disinfectant and secure onto the buckets.
SECTION: Additional Procedures: Tabletop Centrifugation - Swinging Bucket
32. Generally, the buckets do not have their interiors wiped out unless there is a small amount of leakage. In that case, the buckets are thoroughly wiped out with disinfectant and resealed.
SECTION: Additional Procedures: Tabletop Centrifugation - Swinging Bucket
33. It is very important that users check gasket integrity each time the buckets are used.
SECTION: Additional Procedures: Tabletop Centrifugation - Swinging Bucket
34. In case of tube breakage, let the bucket rest in the centrifuge for 10 min, carefully remove the bucket and inspect the seal.


SECTION: Additional Procedures: Tabletop Centrifugation - Micro/High Speed
37. Remove rotor and place into biological safety cabinet. 
SECTION: Additional Procedures: Tabletop Centrifugation - Micro/High Speed
38. Remove aerosol cover. Inspect gasket, replace if necessary.
SECTION: Additional Procedures: Tabletop Centrifugation - Micro/High Speed
39. Wipe down tubes with disinfectant. Place tubes in rotor and replace cover.
SECTION: Additional Procedures: Tabletop Centrifugation - Micro/High Speed
40. Remove rotor from biological safety cabinet with standard removal procedure.
SECTION: Additional Procedures: Tabletop Centrifugation - Micro/High Speed
41. Place into centrifuge, commence run. After run, remove rotor and place into biological safety cabinet.
SECTION: Additional Procedures: Tabletop Centrifugation - Micro/High Speed
42. Remove tubes. Wipe down interior of rotor with disinfectant. Check gasket. Remove rotor from biological safety cabinet with standard procedure. Return rotor to centrifuge.
SECTION: Additional Procedures: Tabletop Centrifugation - Micro/High Speed
43. In case of tube breakage during run: Let rotor rest in centrifuge for 10 min. 

SECTION: Inactivated SARS-Cov-2 TCID determination
46. 
 
Seed Vero E6 cells in complete media (EMEM/10% FBS/1% Pen/Strep) into 96-well microtiter plates (8 x 103cells/well) or 18-48 h before the assay in a BSL2 lab. Optimal cell density is around 2x104 cells/cm2.
SECTION: Inactivated SARS-Cov-2 TCID determination
47. On the day of assay, transfer the Vero E6 cells to the CO2 incubator in the BSL3 lab in a secondary container.
SECTION: Inactivated SARS-Cov-2 TCID determination
48. Prepare 12 x 5-fold serial dilutions. The first dilution should be a 1:1 mixture of warm infection medium (EMEM/3%FBS) and the 2000 cell/ul in SSC cocktail.
SECTION: Inactivated SARS-Cov-2 TCID determination
49. Transfer 50 µL of virus dilution per well onto the Vero E6 cells.

SECTION: Inactivated SARS-Cov-2 TCID determination
50. After 1 h viral adsorption, transfer the secondary container with the culture plate to BSC, remove plate, remove media, and add 150 µL of warm post infection medium at 37 °C to each well.
SECTION: Inactivated SARS-Cov-2 TCID determination
51. Incubate cultures at 37 °C with 5% CO2 for up to 6 days, until CPE is evident.

SECTION: Inactivated SARS-Cov-2 TCID determination
51.1. To monitor cultures for CPE, place a piece of absorbent bench coat paper under a microscope.
SECTION: Inactivated SARS-Cov-2 TCID determination
51.2. Transfer the secondary container with the culture plate from the CO2 incubator to bench, on top of the paper, ensuring that the lid is secured to the secondary container before removing it from the incubator.
SECTION: Inactivated SARS-Cov-2 TCID determination
51.3. Once on the bench, look through the side of the container to see if any liquid has spilled out of the tray.
If there is liquid, move the container to the biosafety cabinet and proceed to decontaminate the tray, and secondary container. 
If there is no spilled liquid, carefully remove the lid to the secondary container and place the tissue culture plate onto the microscope stage. 
 

SECTION: Inactivated SARS-Cov-2 TCID determination
51.4. After examining the plate under the microscope, replace the tray back into the secondary container, secure the lid and return the container to the CO2 incubator, and then carefully open one corner of the lid to the secondary container for air exchange.
SECTION: Inactivated SARS-Cov-2 TCID determination
52. Score positive and negative wells based on CPE observed and calculate TCID50/ml using the Spearman-Karber method. Analyze and validate viral inactivation.
SECTION: Additional Procedures: Basic Biosafety Cabinet Setup
23. 
Biosafety cabinets must be minimally equipped with: 
Overlapping plastic-backed underpads to cover work surface of biological safety cabinet, spritz with disinfectant. 
One container of Vesphene® III disinfectant, with Kimtowel inside the container. 
If pipettes are used, a metal petri dish can (labeled with date and name) containing a bag with a layer of disinfectant inside of it.
Plastic roller bottle or other plastic capped container, half filled with disinfectant.
Biohazard waste bag labeled with name and date, securely attached to inside of biological safety cabinet with tape.
Large waste bucket with biohazard bag. The lid of the bucket must be labeled with name and date.
Several rubber bands.
A 500 ml squirt bottle with disinfectant, markers, pipetmen, and pipette aid.
Add equipment and other supplies as needed.
SECTION: Inactivated SARS-Cov-2 TCID determination
49.1. Wipe the outside of plate with disinfectant and place into a plastic secondary container with lid.
SECTION: Inactivated SARS-Cov-2 TCID determination
49.2. Take the secondary container with the culture plate from the biosafety cabinet according to the protocol "Standard Item Removal Procedure”, and place into the CO2 incubator.
SECTION: Inactivated SARS-Cov-2 TCID determination
49.4. Incubate for 60 min at 37 °C.
SECTION: Inactivated SARS-Cov-2 TCID determination
49.3. Carefully open one corner of the lid to allow air exchange.
SECTION: Inactivated SARS-Cov-2 TCID determination
50.1. Wipe the outside of plate with disinfectant and place into a plastic secondary container with lid.

SECTION: Inactivated SARS-Cov-2 TCID determination
50.3. Carefully open one corner of the lid to allow air exchange.
SECTION: Inactivated SARS-Cov-2 TCID determination
50.2. Take the secondary container with the culture plate from the biosafety cabinet according to the protocol "Standard Item Removal Procedure” and place into the CO2 incubator.
SECTION: Additional Procedures: Standard item removal procedure
25.1. Prior to use, the equipment is placed into the biohazard bag with the cord sticking out of the opening of the bag. Using a twist tie or rubber band to secure the opening around the cord, and place the unit in the biological safety cabinet. If using a vortexer, reinforce the area of the bag over the cup with two pieces of tape.
SECTION: Additional Procedures: Standard item removal procedure
25.4. Wipe hands again and remove the twist tie or rubber band. Keeping one hand on the bag, reach into the bag with the other hand and remove the equipment from the bag and the biological safety cabinet in one movement. Throw bag into waste bag inside of the biological safety cabinet.
SECTION: Additional Procedures: Standard item removal procedure
25.3. Remove hands from the biological safety cabinet, don a fresh pair of outer gloves and re-enter the cabinet.
SECTION: Additional Procedures: Standard item removal procedure
25.2. To remove the equipment, wipe down the area around the opening of the bag and the cord. Wipe down the outer gloves and remove.
SECTION: Additional Procedures: Tabletop Centrifugation - Swinging Bucket
30.1. Wipe down the tubes with disinfectant and place into the buckets.
SECTION: Additional Procedures: Tabletop Centrifugation - Swinging Bucket
30.3. Remove the buckets from the biological safety cabinet using the standard removal procedure.
SECTION: Additional Procedures: Tabletop Centrifugation - Swinging Bucket
30.2. Replace the caps and wipe down the buckets with disinfectant.
SECTION: Additional Procedures: Tabletop Centrifugation - Swinging Bucket
34.1. If it is compromised, consider the tube failure an “out of the cabinet” spill and proceed accordingly.

SECTION: Additional Procedures: Tabletop Centrifugation - Swinging Bucket
34.2. If the gasket is intact, remove the bucket to the biological safety cabinet and clean out tube debris. 
SECTION: Additional Procedures: Tabletop Centrifugation - Swinging Bucket
35. Soak the buckets and lids in a container of disinfectant for 10 min in the biological safety cabinet. Rinse with water.
SECTION: Additional Procedures: Tabletop Centrifugation - Swinging Bucket
36. Then wipe down, replace the covers, and remove from the biological safety cabinet with the standard procedure. Return the bucket to the centrifuge.
SECTION: Additional Procedures: Tabletop Centrifugation - Micro/High Speed
43.1. If you open the centrifuge and it looks like a tube leaked, immediately close the lid and consider this an out of the biological safety cabinet spill. Proceed accordingly.

SECTION: Additional Procedures: Tabletop Centrifugation - Micro/High Speed
43.2. If there is no leakage, then remove rotor, and keeping it level; remove it to the biological safety cabinet.

SECTION: Additional Procedures: Tabletop Centrifugation - Micro/High Speed
44. In the biological safety cabinet, take off the cover of the rotor and remove all tubes and debris. Soak rotor and cover in a container of disinfectant for 10 min in the biological safety cabinet.
SECTION: Additional Procedures: Tabletop Centrifugation - Micro/High Speed
45. Rinse with water. Then wipe down, replace the cover, and remove.
SECTION: Additional Procedures: Standard item removal procedure
24.1. The item is wiped down with disinfectant. 
SECTION: Additional Procedures: Standard item removal procedure
24.2. Wipe the outer pair of your gloves with disinfectant, remove gloves, and throw them into red waste bag in the biological safety cabinet.
SECTION: Additional Procedures: Standard item removal procedure
24.3. Gloved hands can then be removed from the cabinet; a fresh pair of outer gloves is put on and you then re-enter the biological safety cabinet.
SECTION: Additional Procedures: Standard item removal procedure
24.4. Wipe down gloves and the surface of the item with disinfectant, then remove item from the cabinet.