MGH Harvard SenNet Processing murine lung 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.

Pipet 1ml digestion solution on the lid of a 10cm dish. Place the lung in it. Using a razor blade, "mince" the lung, holding one tip with the forceps.

Pipet the sample into 4ml of the digestion solution / lung (8ml per mouse) and incubate at 37*C for 30min with rocking (in a 15ml falcon tube).

Use a Pasteur pipet to triturate suspension until tissue is fully dissociated.

Pass the suspension through a 100 μm cell strainer into 50-mL tubes and use 10 mL of DPBS to pass through the strainer to wash it.

Centrifuge at 1000 × g for 5 min at 4°C, discard the supernatant.

Add 1-2 mL of 1× RBC lysis buffer to resuspend the pellet and incubate for 5 min at 20°C–26°C.

Add 10 mL of DPBS to stop the lysing,

Centrifuge at 1000 × g for 5 min at 4°C, discard the supernatant.

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

Count two samples each per lung and note down the exact number of cells. This information is important for scRNA library prep. We routinely recover 8-10 million cells / mouse with this protocol.

Split lung samples into 100,000 cell aliquots, according to the cell counts determined above.

Keep one aliquot on ice for the scRNA-seq sample.

For the remaining aliquots, spin down at 1000 × 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