Single nuclei isolation of cardiac samples and processing on the 10X Genomics platform

Samples were processed independent of disease, genotype, or control status. Nuclei isolation and library preparation was performed at the Harvard Medical School, Imperial College London, and the Max-Delbrück Center for Molecular Medicine as previously reported (1, 2). Isolated nuclei from flash-frozen tissue were visually inspected under the microscope to assess nuclei integrity and automatically counted using a Countess II (Life Technologies). Nuclei suspension was loaded on the Chromium Controller (10X Genomics) with targeted nuclei

recovery of 5,000–10,000 per reaction (Table S2). 3′ gene expression libraries were prepared according to the manufacturer’s instructions of the v3 Chromium Single Cell Reagent Kits (10X Genomics). Quality control of final library cDNA was done using Bioanalyzer High

Sensitivity DNA Analysis (Agilent) and the KAPA Library Quantification kit. Libraries were sequenced on an Illumina HighSeq 4000 or NovaSeq with a targeted read number of 30,000-50,000 reads per nucleus (Table S2).

Protocols are published:

1. https://www.nature.com/articles/s41586-020-2797-4
2. https://currentprotocols.onlinelibrary.wiley.com/doi/full/10.1002/cpz1.132
3. https://www.protocols.io/view/single-cell-and-single-nuclei-analysis-human-heart-x54v98pkml3e/v1