Aug 21, 2023
MGH Harvard SenNet Processing murine trachea for paired single cell RNA-seq and mass spec
- 1Massachusetts General Hospital;
- 2Harvard Medical School
Protocol Citation: gshipkovenska Shipkovenska 2023. MGH Harvard SenNet Processing murine trachea for paired single cell RNA-seq and mass spec. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbxx93lpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 21, 2023
Last Modified: August 21, 2023
Protocol Integer ID: 86757
Funders Acknowledgement:
NIH
Grant ID: GRANT13844270
Abstract
Protocol for obtaining single cell suspension of murine trachea.
Materials
PBS - Phosphate-Buffered Saline (10X) pH 7.4, RNase-free (Thermo Fisher
Scientific; cat. no: AM9625)
1x PBS Buffer for washes and cell suspension
5ml Phosphate-Buffered
Saline (10X) pH 7.4, RNase-free (Thermo Fisher Scientific; cat. no: AM9625).
45ml Invitrogen water
Important: Do not use DPBS or other PBS buffers.
0.5% BSA in 1x PBS solution
Dissolve 0.250mg BSA in 1xPBS solution above.
DNase I (50,000x) 50,000 U/ml
Papain Stock Solution (2X)
40u/ml Papain (lyophilized, Worthington-Biochem, cat# LS001220) in EBSS (has Mg
and Ca).
Dissolve 120 mg papain in 50 ml EBSS by rocking at room temp for 20min. Filter using
syringe filters in the hood and aliquot in the hood in 1 ml tubes. Freeze at -20*C.
Activation Stock Solution (2X)
0.067 mM beta-mercaptoethanol, 1.1 mM EDTA, 5.5 mM Cysteine-HCl, in EBSS.
Combine:
110ul of 500 mM EDTA
66ul 50 mM beta-mercaptoethanol (deli fridge in tissue culture room)
0.048g ysteine-HCl.H2O (powder above Brian's bench).
50ml EBSS
(Changes color to orange)
Freeze at -20*C.
RBC Lysis buffer (commercial, ThermoFisher Scientific, 00-4333-57)
Enzyme dissociation mix #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).
Note:
Can scale down from 5ml for the number of tracheas processed.
100% FBS
Thaw in advance in water bath.
DMSO
Dissection
Dissection
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.
Single cell suspension
Single cell suspension
Incubate each trachea in 500ul enzyme dissociation mix #1 for 30min with rotation at 37*C.
Enzyme dissociation mix #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
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 cells with heamocytometer
Count cells with heamocytometer
Count two samples each per trachea. We routinely recover 250,000-350,000 cells with this protocol.
Prepare samples for scRNA-seq and scMS
Prepare samples for scRNA-seq and scMS
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.
scRNA library preparation
scRNA library preparation
Prepare according to manufacturer's protocol: 
CG000204_ChromiumNextGEMSingleCell3_v3.1_Rev_D.pdf3.7MB
CG000204_ChromiumNextGEMSingleCell3_v3.1_Rev_D.pdf3.7MB