MGH Harvard SenNet Processing murine trachea for paired single cell RNA-seq and mass spec

Euthanize mice by CO2 method.

Perfuse lungs via right ventricle.

Dissect out trachea and lungs and place in HBSS on ice.

Clean up the tracheas under the dissection microscope.

Bi-sect the tracheas along the ventral side.

Incubate each trachea in 500ul enzyme dissociation mix #1 for 30min with rotation at 37*C.

Enzyme dissociation mix #1 1

1ml of papain

1ml of activation solution

20ul Dnase I

Cut off the tip of p1000 tip and pipet trachea up and down 10 times. Should get stringy.

Spin down for 5 min at 2000g. This will pellet both single cells and the husk.

Using a pipet (not the vacuum), remove as much supernatant as possible without being a hero.

Resuspend cells and husk in 500ul enzyme mix #2 per trachea for 20 minutes at 37C with rocking.

Enzyme dissociation mix #2 2

25ul of 70kU/ml Collagenase I (in DMEM/ringers)

25ul of 50kU/ml hyluronidase (in DMEM/ringers)

50ul of 7.5kU/ml DNAse (in DMEM/ringers)

120ul of 2.5U/ml dispase (in DMEM/ringers)

400ul of 2X papain (40 u/ml) (in EBSS)

QC to 5 ml of DMEM (has Ca and Mg, so good to use).

Cut off the tip of p1000 tip and pipet trachea up and down 10 times. Husk will be mostly gone.

Spike in 55ul FBS for 10% FBS final concentration to inactivate papain and mix well.

Strain cells through 100um strainer.

Centrifuge at 8000 × g for 2.5 min, discard the supernatant.

Add 300 uL of RBC lysis buffer per trachea and incubate for 60s on the bench.

Add 1 mL of EBSS to stop the lysing, centrifuge at 8000 × g for 2.5 min, discard the supernatant.

Resuspend with 200 uL of 0.5% BSA in PBS. Keep on ice.

Count two samples each per trachea. We routinely recover 250,000-350,000 cells with this protocol.

Split each tracheal sample into 50,000 cell aliquots, according to the cell counts determined above.

Keep one aliquot on ice for Andrew to pick up. This is the scRNA-seq sample.

For the remaining 3-4 aliquots, spin down at 8000 × g for 5 min, discard the supernatant and resuspend in 90% FBS, 10% DMSO and freeze at -80*C. These are the scMS samples.

Prepare according to manufacturer's protocol: CG000204_ChromiumNextGEMSingleCell3_v3.1_Rev_D.pdf