TITLE: Cell dissociation from nasal and bronchial brushings with cold-active protease for single-cell RNA-seq
AUTHORS: Laure-Emmanuelle Zaragosi, Pascal Barbry

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

[STEPS]
?. Perform nasal brush biopsy in the nasal cavity in the inferior nasal conca zone (UBERON_0005922) (to be performed by a medical doctor)
?. Cut the cytology brush and place it in a 5 mL eppendorf tube containing 1 mL of ice-cold dissociation buffer.
1 ml
PREPARATION OF DISSOCIATION MIX (Fresh at each experiment)Ingredients:- Hypothermosol- Protease from Bacillus Licheniformis (100 mg/mL stock solution in PBS)- EDTA 10 mMFor 1 mL of dissociation mix add:- 850 microlitres of Hypothermosol- 100 microlitres of protease (Final concentration:10 mg/mL)- 50 microlitres of EDTA (Final concentration: 0.5 mM)
?. Shake the brush into the buffer to put cells in suspension then spin the tube for 2 min at 150g to remove residual cells from the brush.
?. 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.If storage and/or transportation has been performed for at least 60 min, skip the incubation step, only perform trituration steps.
[Incubation]
[Trituration]
?. Discard the cytology brush and observe cells under an inverted microscope.
?. Inactivate protease by adding 200 µL of Inactivation buffer (HBSS containing 2% BSA)
[Inactivation buffer]
Prepare Inactivation buffer: HBSS : 40 mL10% BSA stock: 10 mL
?. Spin at 400g for 5 min at 4°C
?. Discard supernatant leaving 10 µL of residual liquid on the pellet.
?. 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.
?. If RBC content iis lower than 50%, go directly to step 18.Perform RBC lysis: add 1.8 mL of Ammonium Chloride 0.8% to 200 µL of cell suspension (9 volumes), transfer in a 5 mL eppendorf.
[Ammonium Chloride 9 vol. for 1 cell vol.]
?. Incubate on ice for 10 min.
[RBC lysis]
?. Add 400 µL of Inactivation buffer
[Inactivation buffer]
?. Spin at 400g for 5 min at 4°C
?. Resuspend in 200 µL of wash buffer (HBSS + 1% BSA)
Prepare Wash buffer: HBSS : 20 mLHBSS/BSA 2%: 20 mL
?. Spin at 400g for 5 min at 4°C
?. Resuspend in 250 µL of wash buffer and monitor correct RBC lysis
[wash buffer]
?. Discard supernatant leaving 10 µL of residual liquid on the pellet.
?. Add 1 mL of wash buffer
[wash buffer]
?. Discard supernatant leaving 10 µL of residual liquid on the pellet.
?. Resuspend in 500 µL of wash buffer
[wash buffer]
?. Discard supernatant leaving 10 µL of residual liquid on the pellet.
?. Resuspend in 50 µL of resuspension buffer (HBSS + 0.05% BSA).
[Resuspension buffer]
Prepare Resuspension buffer: HBSS : 39 mLHBSS/BSA 2%: 1 mL
?. Mix 10 µL of cells with 10 µL of cell counting solution (HBSS with 20 µg/mL Hoechst 33342 and NucGreen). Incubate for 1 min at room temperature.
Preparation of cell staining reagent: - HBSS: 500 µL- Hoechst 33342 (10 mg/mL): 1 µL- NucGreen™ Dead 488 ReadyProbes™ Reagent: 1 drop
?. Filter cell suspension through Flowmi cell strainer https://www.youtube.com/watch?v=taS1BuTnvds
?. Add 500 µL of wash buffer to filtered cells.
[wash buffer]
?. Count with Countess automated cell counter using both sides of chambers. Monitor the percentage of nucleated cells (Hoechst +) and dead cells (GFP+).
Countess report after NucGreen and Hoescht33342 staining
Countess bright field image after NucGreen and Hoescht33342 staining
Countess Dapi image after NucGreen and Hoescht33342 staining
?. If cells are processed directly after brushing, use DPBS instead of hypothermosol and go directly to step 4.If transportation or storage is necessary: place tube on ice or in polystyrene box containing ice packs. Brushings can be stored for 60 min in dissociation buffer.
?. Adjust concentration to a range of 700 to 1000 cells/µL (with wash buffer) for 10X Chromium. Monitor final cell concentration.