Customer Developed Protocol

Preparation of non-myocyte cardiac single cellsuspensions
Contributed by:Galen Squiers & Alex Pinto, Pinto Lab, The Jackson Laboratory, Bar Harbor,USA

CUSTOMER DEVELOPED PROTOCOL
Preparation of non-myocyte cardiac single cell suspensions

Contributor Research Profile
Nadia Rosenthal, Ph.D., F.Med.Sci is a scientific director and professor with the Jackson Laboratory for Mammalian
Genetics. Rosenthal’s research uses mammalian genetics to explore the embryonic development of heartand skeletal muscle and the regeneration of adult tissues. She focuses on muscle and cardiac developmental
genetics and the role of growth factors, stem cells and the immune system in tissue regeneration. She is a global
leader in the use of targeted mutagenesis in mice to investigate muscle development, disease and repair, and is aparticipant in EUCOMM, the European Conditional Mouse Mutagenesis Program, where she coordinates theselection and production of new Cre driver strains for the international mouse genetics community.
Link to the lab: https://www.jax.org/research-and-faculty/research-labs/the-rosenthal-lab

Reference
This protocol was used in:
Skelly et al.: Single cell transcriptional profiling reveals cellular diversity and intercommunication in themouse heart. Cell Reports, Jan. 16, 2018https://www.ncbi.nlm.nih.gov/pubmed/29346760
Publication Press Release: https://www.jax.org/news-and-insights/2018/january/new-census-of-mouse-heartcells

10x Genomics Products
10x Chromium™ Single Cell Gene Expression Solution - https://www.10xgenomics.com/single-cell/

Notices
THIS PROTOCOL IS NOT SUPPORTED BY 10X GENOMICSThis Customer Developed Protocol is provided for general information only and is not directly supported,endorsed, or certified by 10x Genomics. 10x Genomics gives no warranties and makes no claims about theprovided protocol.
For questions or to share protocol adjustments, please visit the 10x Community at community.10xgenomics.com

Customer Developed Protocol

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CUSTOMER DEVELOPED PROTOCOL
Preparation of non-myocyte cardiac single cell suspensions

Required Buffers and Reagents
1. Perfusion Buffer (1 × DPBS (Gibco, cat#: 14190250), supplemented with 0.8mM CaCl2)
2. Washing buffer (1 × HBSS (Gibco, cat#: 14065056))
3. Digestion Buffer
2 mg/ml collagenase type IV (Worthington Biochemical CLS-4)
1.2 U/ml Dispase II (Sigma-Aldrich, cat#: 255-914-4)
0.8 mM, 1 × DPBS

4. 70 µm cell strainer (Fisher Scientific, cat#: 22-363-548)
5. FACS buffer (1 × HBSS, 2% fetal bovine serum (Gibco, cat#: 26140079))

Protocol Notes•

Isolated hearts are usually collected and in a well of a 24 well dish (placed on ice) with ~2 ml of Perfusion
Buffer. Usually all hearts are gathered, trimmed, and weighed (optional) before proceeding to Step 3.

•Tissue mincing is usually conducted on 60mm tissue culture dish.

•At the first trituration, it is common for tissue to get stuck while being drawn up the pipette tip.

•This and the following step removes most of the cardiomyocyte/tissue debris. Cells form a pellet whiledebris remains in the supernatant.

ProtocolNOTE: All samples and reagents are kept on ice or at 4 °C except during the tissue dissociation step (Step 5).
1. Mice were euthanized by CO2 asphyxiation and sprayed with 70% ethanol to minimize dispersal of fur.
2. Hearts were accessed by dissection of anterior thoracic wall and mouse tissue perfused for 10 min with
4°C Perfusion Buffer via left ventricle using a peristaltic pump (~2 ml/min).
3. Hearts were removed and trimmed to isolate ventricles and separate atria and valves (see Note 1).
4. Ventricles were minced into ~1 mm cubes using surgical scissors (see Note 2) and placed in 5 ml tubes
containing 3 mL Digestion Buffer.
5. Digestion Buffer/tissue mixture was incubated at 37° C for 45 min in a water bath, and triturated every
15 min using a 1000 µl pipette (see Note 3). At the 15 and 30 min time points, Digestion buffer/tissuemixtures were triturated by pipetting up and down for 15 repetitions. The final trituration was done
identically at the 45 min time point, except Digestion Buffer/tissue mixture was pipetted up and downfor 30 repetitions.
6. Following final trituration, Digestion Buffer/tissue mixture was filtered using a 70 µm cell strainer into a
15 ml tube containing ~10 ml Perfusion Buffer

Customer Developed Protocol

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CUSTOMER DEVELOPED PROTOCOL
Preparation of non-myocyte cardiac single cell suspensions
7. The 15 ml tube was topped-up with Perfusion Buffer before being centrifuged at 200 RCF for 20 min at
4° C with breaks disengaged (see Note 4).
8. The supernatant was aspirated and the cell pellet resuspended in 15ml Washing Buffer before being
centrifuged again at 200 RCF for 20 min at 4° C with breaks disengaged.
9. After aspirating the supernatant, the cell pellet was resuspended in FACS Buffer before subsequent
processing/analysis.

THIS PROTOCOL IS NOT SUPPORTED BY 10X GENOMICSThis 10x Community Customer Developed Protocol is provided for general information only andis not directly supported, endorsed, or certified by 10x Genomics. 10x Genomics gives no warranties and makes
no claims about the provided protocol.
For questions or to share protocol adjustments, please visit the 10x Community at community.10xgenomics.com

Customer Developed Protocol

COMMUNITY.10XGENOMICS.COM