TITLE: Cell dissociation from nasal, bronchial and tracheal brushings with cold-active protease for single-cell RNA-seq
AUTHORS: Kaylee Worlock, Masahiro  Yoshida, Kerstin Meyer, Marko  Z. Nikolić

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This modified protocol provides details on the cell dissociation that should be performed to obtain single-cell suspensions from nasal, tracheal and bronchial epithelium brushings.</div><div class = "text-block">Cell dissociation is performed at 4°C to avoid gene expression alterations and maximize viability for scRNAseq.</div><div class = "text-block">The typical cell number recovery is 200,000-300,000 per nasal brushing and 10,000-30,000 per tracheal/bronchial brushing .Cell suspensions are suitable for single-cell RNA-sequencing protocols with viability generally >70%.</div></div>

[STEPS]
?. [Sample Collection and processing]
Perform nasal brush biopsy in the nasal cavity in the inferior nasal conca zone (UBERON:0005922) using cytological sampling brushes (CY1050). Tracheal and bronchial brush biopsies were collected using single-use cytology brushes (BC-202D-2010) from the tracheal mucosa (UBERON:0000379) and bronchial mucosa (UBERON:0000410). Brushings were performed by a medical doctor.
?. [Sample Collection and processing]
Transfer brush (or brushes if multiple tracheal or bronchial brushes were taken) into a new 15 mL falcon tube containing 1 mL of dissociation mix (Dissociation tube) and leave on ice to help collect any remaining cells still attached to the brush.
1 mL
4 °C
?. [Sample Collection and processing]
Gentle re-suspend the cell pellet in remaining supernatant in each 15 mL falcon tube and transfer into Tube A.
If multiple nasal brushes from the same patient were collected (one from each nostril) at this point the cells were all pooled into one tube (Tube A).
?. [Sample Collection and processing]
Spin Tube A and Dissociation tube (with brush/brushes still inside) for 5 mins at 400g.
[ Spin at 400xg (4 °C)]
?. [Sample Collection and processing]
Incubate cells on ice for 30 min, with gentle trituration with needles 5 times every 5 min. Use needle with decreasing sizes from 21G to 23G.
[Incubation]
[Trituration]
4 °C
?. [Sample Collection and processing]
Inactivate 1 mL of dissociation mix by adding 200 µL of Inactivation buffer (HBSS containing 2% BSA)
[Inactivation buffer]
?. [Sample Collection and processing]
Spin at 400g for 5 min at 4°C
[ Spin at 400xg (4 °C)]
?. [Sample Collection and processing]
Discard supernatant leaving 200 µL of residual liquid on the pellet.
?. [Sample Collection and processing]
Spin at 400g for 5 min at 4°C
[ Spin at 400xg (4 °C)]
?. [Sample Collection and processing]
Discard supernatant leaving 10 µL of residual liquid on the pellet.
?. [Sample Collection and processing]
Resuspend in 1  mL of wash buffer
[wash buffer]
?. [Sample Collection and processing]
Spin at 400g for 5 min at 4°C
[ Spin at 400xg (4 °C)]
?. [Sample Collection and processing]
Discard supernatant leaving 10 µL of residual liquid on the pellet.
?. [Sample Collection and processing]
Filter 500 µL of cell suspension through Flowmi cell strainer (repeat)  https://www.youtube.com/watch?v=taS1BuTnvds
We filter using 2 x 500 µL steps and try to avoid taking up any obvious solid mucous clumps to remove the risk of blockages in the Flowmi and losing cells.
?. [Sample Collection and processing]
Resuspend in 20-50 µL of resuspension buffer depending on the size of the pellet (HBSS + 0.05% BSA).
[Resuspension buffer]
?. [Sample Collection and processing]
Adjust concentration to 1000 cells/µL (with resuspension buffer) for 10X Chromium. Monitor final cell concentration.
?. [Sample Collection and processing]
Carefully cut the cytology brush and place straight into a 15mL falcon tube (Tube A) containing 4 mL of cold transport media and store at 4 degrees Celsius. Where possible samples should be processed immediately after collection. However, where multiple samples were collected, brushes were stored at 4 degrees Celsius for up to 2-3 hours to allow them to be processed together.
4 mL
Collecting multiple brushes from the same donorIf multiple tracheal or bronchial brushes were collected from the same donor at this point they were collected in the same 15 mL falcon tube due to size. Whereas if two nasal brushes from the same donor were collected (one from each nostril) these were placed in their own 15 mL falcon tube and the cells collected together later in step 8.
?. [Sample Collection and processing]
Working in a CL2 hood on ice shake the 15 mL falcon tube (Tube A) containing the cytology brush vigorously (~30 seconds) to help dislodge cells and mucous. Place the tube back on ice for 1 min, allowing the aerosols to settle, before using a sterile set of tweezers to carefully transfer the brush from Tube A into a new 15 mL falcon tube (Tube B) containing 4 mL HBSS. Repeat shaking step, transferring the brush to a new 15 mL flacon tube containing 4 mL HBSS each time (e.g Tube C and Tube D etc) until no mucus or cells can be seen on the brush and/or to be coming off into the HBSS.
If samples from multiple different patients are being processed at the same time be sure to wash the tweezers with 70% ethanol and dry throughly between samples to help avoid cross contamination.
?. [Preparation of reagents and tubes (before starting): ]
PREPARATION OF DISSOCIATION MIX (Fresh at each experiment)Ingredients:- Hypothermosol- Protease from Bacillus Licheniformis (100 mg/mL stock solution in PBS)- EDTA 10 mMPrepare 1 mL per sample:- 850 microlitres of Hypothermosol- 100 microlitres of protease (Final concentration:10 mg/mL)- 50 microlitres of EDTA (Final concentration: 0.5 mM)Aliquote 1 mL of dissociation mix in a 15 mL falcon tube for every sample (Dissociation tube).
PREPARATION OF TRANSPORT MEDIA (STORED FOR UP TO ONE MONTH)Ingredients:- αMEM (Gibco; 22561-01)- Pen Strep (Gibco; 15070)(5,000 units/mL Penicillin and 5,000 μg/mL Streptomycin)- Amphotericin B (Fisher Scientific; 10746254) (250 μg/mL)- Gentamicin (Gibco; 15710) (10mg/mL)For Transport Media supplement aMEM media bottle with: - 5 mL Pen Strep (Finial concentration: 50 units/mL Penicillin and 50 μg/mL Streptomycin)- 0.5 mL Gentamicin (Finial concentration; 10 ng/mL)- 0.5 mL Amphotericin B (Finial concentration; 250 ng/mL)Aliquote 4 mL of Transport media  in a 15 mL falcon tube for every sample (Tube A).
PREPARATION of INACTIVATION, WASH and RESUSPENSION BUFFERS:Prepare stocks of 10% BSA in HBSS and aliquot and store at -20 °C (filtered; 22mm filter).Prepare inactivation buffer; 2% BSA/HBSS (aliquot and store @ -20 °C or @ 4°C for 1 week)-40 mL HBSS+10 mL 10% BSA stock.Prepare wash buffer; 1% BSA/HBSS (aliquot and store @ -20 °C or @ 4°C for 1 week)- 20 mL HBSS + 20 mL 2% BSA/HBSS.Prepare resuspension buffer; 0.05% BSA/HBSS (aliquot and store @ -20 °C or @ 4°C for 1 week)-1 mL 2% BSA/HBSS + 39 mL HBSS
?. [Sample Collection and processing]
Carefully remove supernatant from each tube, leaving ~0.5-1 mL.
?. [Sample Collection and processing]
Spin all 15 mL falcon tubes used in Step 4. (Tube A, B , C and D ect.)  for 5 mins at 400g.
[ Spin at 400xg (4 °C)]
?. [Sample Collection and processing]
Discard brush/brushes from Dissociation Tube and carefully remove the supernatant from Tube A.
Try to remove as much of the supernatant as you can from Tube A, but sometime if the sample contains a lot of mucous this can be difficult, particularly if some of the mucous is seen to be floating. If it is particularly challenging you can try briefly re-spinning the sample at 400g for 2 mins at 4 °C and leaving slightly more supernatant in the tube.
?. [Sample Collection and processing]
Re-suspend any cells that might have collected first in the bottom of the Dissociation tube in the dissociation mix before transferring it to Tube A using a P100 pipette and gentle re-suspending the cell pellet.
?. [Sample Collection and processing]
Add 9 mL of Inactivation buffer
[PBS]
?. [Sample Collection and processing]
Incubate at room temperature (RT)  for 3 min.WARNING: Be careful not to exceed 3 min or you will start to lyse your non-RBCs.
[RBC lysis]
4 Room temperature
?. [Sample Collection and processing]
If RBC content appears lower than 50% or the pellet is hardly visible, go directly to step 21. If the cell pellet appears bloody by eye (>40 % red), and/or under the microscope > 50% of the cells observed are RBCs perform RBC lysis: add 1mL of 1x RBC lysis buffer (Invitrogen, #004333-57) and gently re-suspend the cell pellet .
[RBC lysis buffer at RT]
Observe cells under an inverted microscope to evaluate red blood cells (RBC) content. RBC content is better evaluated using an automated cell counter such as Countess, after addition of Hoechst 33342 to an aliquot of the cell suspension to discriminate nucleated cells from non-nucleated cells.
?. [Sample Collection and processing]
Preform cell count and calculate viability using trypan blue (Thermo Fisher; 15250061) and a hemocytometer.
Viability ideally > 70% for use with 10x.
?. [Sample Collection and processing]
Briefly re-spin tubes again to collect any residual liquid left on the side of the tubes (to ensure volume is accurate) and discard supernatant leaving 10 µL of residual liquid on the pellet.
[ Spin at 400xg (4 °C)]