Mar 04, 2025
Harvesting cells for single-cell RNA-sequencing
- 1Northwestern University
Protocol Citation: Alexander Misharin 2025. Harvesting cells for single-cell RNA-sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxwjbov8j/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: March 03, 2025
Last Modified: March 04, 2025
Protocol Integer ID: 123732
Abstract
Protocol for extracting samples from mouse lung tissue and then harvesting cells to be used in scRNA-seq.
Guidelines
Research involving animals must be conducted according to internationally-accepted standards and should always have prior approval from the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Protocol materials
Fetal Bovine Serum (FBS)ATCCCatalog #30-2020
Step 10
Acridine orangeFisher ScientificCatalog #AC423340010
Step 11
HBSSGibco - Thermo Fisher ScientificCatalog #14060040
Step 2
BD Pharm LyseBD BiosciencesCatalog #555899
Step 9
DMEMInvitrogen - Thermo Fisher
Step 10
Propidium iodide
Step 11
Pentobarbital sodiumMerck MilliporeSigma (Sigma-Aldrich)Catalog #Y0002194
Step 1
DispaseCorningCatalog #354235
Step 3
deoxyribonuclease I (DNase I, 100 U/ml)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D 4263
Step 3
Mice were euthanized with Pentobarbital sodiumMerck MilliporeSigma (Sigma-Aldrich)Catalog #Y0002194
The chest cavity was opened and lungs were perfused through the right ventricle with 10 mL HBSSGibco - Thermo Fisher ScientificCatalog #14060040
The lungs were removed and, using a30 g needle, infused with 1 mL DispaseCorningCatalog #354235 with deoxyribonuclease I (DNase I, 100 U/ml)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D 4263
The lungs were incubated at room temperature with gentle agitation for 00:45:00
The lungs were gently teased into small (1-2 mm ) fragments
The lungs were incubated in a digestion buffer for another 00:15:00
The resulting suspension was passed through a 70 µm cell strainer and washed with DMEM supplemented with 5% FBS
The resulting suspension was pelleted by centrifugation
Erythrocytes were lysed using BD Pharm LyseBD BiosciencesCatalog #555899
The resulting single-cell suspension was kept in DMEMInvitrogen - Thermo Fisher and Fetal Bovine Serum (FBS)ATCCCatalog #30-2020 and passed through a
Equipment
40 mm cell strainer
NAME
Falcon
BRAND
352340
SKU
LINK
two times
Cells were counted using a
Equipment
K2 Fluorescent Cell Counter
NAME
Cell Counter
TYPE
Cellometer
BRAND
CMT-K2-MX-150
SKU
LINK
with nucleic acid binding dyes Acridine orangeFisher ScientificCatalog #AC423340010 to calculate total number of nucleated cells and Propidium iodideContributed by users to count dead cells