GSM9456130: snRNA-seq, donor, D1192; Homo sapiens; RNA-Seq
Frozen myocardial tissue from left ventricular assist device (LVAD) cores or the corresponding region from the apex of explanted donors were minced with a razor blade and dissociated with a plastic pestle in Chromium Nuclei Isolation Kit Lysis Buffer (10X Genomics, Pleasanton, CA). Samples were incubated on ice for 10 minutes, and the tissue suspension was pipetted onto the Nuclei Isolation Column and Collection Tube and centrifuged at 16,000 rcf for 20 seconds at 4°C. Flow-through in the Collection Tube was centrifuged again at 1,000 rcf for 3 minutes to pellet the nuclei and resuspended in 300 μl of Wash and Resuspension Buffer before staining with 1 μl  DRAQ5 (5 mM solution, ThermoFisher Scientific) and incubating on ice for 10 minutes. DRAQ5+ nuclei were sorted into Nuclei Wash Buffer on a BD FACS Melody (BD Biosciences) using a 100 μm nozzle. Recovered nuclei were centrifuged again under the above parameters and were gently resuspended in Nuclei Wash Buffer to a target concentration of 1,000 nuclei μl−1. Nuclei were counted on a hemocytometer and concentration was adjusted as necessary. Following flow cytometry, sorted DRAQ5+ nuclei were centrifuged at 2,000 rcf for 5 minutes at 4°C, and nuclei were resuspended in 100 µl 1X PBS, prior to loading into the ICELL8 cx system. Libraries were prepared using the manufacturers' instructions- SMART-Seq Pro (ICELL8 cx Single-Cell System User Manual kit (Takara Bio, San Jose, CA)-and sequenced on a NovaSeqX (Illumina) instrument following manufacturers' guidelines: 151 X 8 X 8 X 151 [read1 X i7 index X i5 index X read2].