TITLE: Cell dissociation from airway biopsies 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 airway biopsies.</div><div class = "text-block">Biopsies may come from tracheal, bronchial or nasal epithelium.</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 40 000 cells for one biopsy.</div><div class = "text-block">Cell suspensions are suitable for single-cell RNA-sequencing protocols.</div></div>

[STEPS]
?. Perform bronchial biopsy at the desired level of the airways (to be performed by a medical doctor)
?. Put the biopsy in 1 mL DPBS in a well of a 6-well plate, observe aspect, and then transfer into 1 mL of ice-cold dissociation buffer in a 1.5 mL eppendorf tube. Use wide-bore 1 mL pipet tip for all biopsy transfers.
1 ml
PREPARATION OF DISSOCIATION MIX (Fresh at each experiment)Ingredients:- PBS- Protease from Bacillus Licheniformis (100 mg/mL stock solution in PBS)- EDTA 10 mMFor 1 mL of dissociation mix add:- 850 microlitres of PBS- 100 microlitres of protease (Final concentration:10 mg/mL)- 50 microlitres of EDTA (Final concentration: 0.5 mM)
?. Incubate cells on ice for 90 to 120 min after mincing, with gentle trituration with needles 5 times every 5 min. Use needle with decreasing sizes from 21G to 23G.
[Incubation]
[Trituration]
?. 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 900 µL of Ammonium Chloride 0.8% to 100 µL of cell suspension (9 volumes).
[Ammonium Chloride 9 vol. for 1 cell vol.]
?. Incubate on ice for 10 min.
[RBC lysis]
?. Add 200 µL of Inactivation buffer
[Inactivation buffer]
?. Spin at 400g for 5 min at 4°C
?. Resuspend in 100 µL of wash buffer (HBSS + 1% BSA)
[wash buffer]
Prepare Wash buffer: HBSS : 20 mLHBSS/BSA 2%: 20 mL
?. Spin at 400g for 5 min at 4°C
?. Resuspend in 100 µL of wash buffer and monitor correct RBC lysis under microscope
[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 40 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+).
?. If transportation or storage is necessary: place tube on ice or in polystyrene box containing ice packs. Biopsies 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.
?. Carefully remove biopsy from the dissociation buffer, with a wide-bore 1 mL pipet tip and place in a 100 mm petri dish, taking as little dissociation buffer as possible. Mince with a scalpel equipped of a 10 blade. Drag the biopsy out of the liquid and mince very carefully into the smallest possible pieces. With a wide-bore pipet tip, transfer back the minced biopsy with a small volume of dissociation buffer. Rince the petri dish with dissociation buffer, at the location of biopsy mincing to recover as many cells as possible.