GSM9456141: snRNA-seq_NICM, T1342; 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 10X Chromium system. After manually counting nuclei using Trypan Blue, based on the concentration, samples were loaded at a volume to target 20,000 nuclei. Libraries were prepared using the manufacturers' instructions-10X 3' v4 GEM-X (Chromium GEM-X Single Cell 3' Reagent Kits v4 Document Number CG000731 Rev B August 2024) or 10X 5' v3 GEM-X (Chromium GEM-X Single Cell 5' Reagent Kits v3 Document Number CG000733 Rev A March 2024) kits (10X Genomics)- and sequenced on a NovaSeqX (Illumina, San Diego, CA) instrument following manufacturers' guidelines: 28 X 10 X 10 X 90 [read1 X i7 index X i5 index X read2].