Jun 20, 2023
MGH Harvard SenNet Processing murine lung for paired single cell RNA-seq and mass spec
- 1Massachusetts General Hospital;
- 2Harvard Medical School
- Cellular Senescence Network (SenNet) Method Development Community
Protocol Citation: gshipkovenska Shipkovenska 2023. MGH Harvard SenNet Processing murine lung for paired single cell RNA-seq and mass spec. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwd6e9lmk/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: June 20, 2023
Last Modified: June 20, 2023
Protocol Integer ID: 83738
Funders Acknowledgements:
NIH
Grant ID: GRANT13844270
Abstract
Protocol for obtaining single cell suspension of murine lung.
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
5mlPhosphate-Buffered Saline (10X) pH 7.4, RNase-free (Thermo Fisher Scientific; cat. no: AM9625).
45ml Invitrogen water
0.5% BSA in 1x PBS solution
Dissolve 0.250mg BSA in 1xPBS solution above.
DNase I (50,000x) 50,000 U/ml
Liberase Stock Solution (2.5mg/ml, 13 Wunsch units/ml)
To one 5mg vial, add 2ml cold Invitrogen ultrapure water. Mix on ice by periodically stirring. Can rock on rocking platform at 4*C for maximum 30min. Aliquot in 50-100ul aliquots (~40) and freeze at -20*C. Do not freeze thaw.
Enzyme master-mix
| Origin concentration | Volume for 4ml (per lung) | ||
| Liberase TM (Roche) (Cat# 05 401 119 001) | 2,500ug/mL | 160ul | |
| DnaseI | 50,000U/ml | 5ul | |
| RPMI 1640 (has Mg and Ca) | 3.640 mL | ||
| FBS | 200ul |
RBC Lysis buffer (commercial, Thermo Fisher Scientific, 00-4333-57)f
100% FBS
DMSO
Dissection
Dissection
Euthanize mice by CO2 method.
Perfuse lungs via right ventricle.
Dissect out trachea and lungs and place in HBSS on ice.
Single cell suspension
Single cell suspension
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 cells with heamocytometer
Count cells with heamocytometer
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.
Prepare samples for scRNA-seq and scMS
Prepare samples for scRNA-seq and scMS
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.
scRNA library preparation
scRNA library preparation
Prepare according to manufacturer's protocol:
CG000204_ChromiumNextGEMSingleCell3_v3.1_Rev_D.pdf Protocol references