GSM7072864: TWCM-371-NR-pre; Homo sapiens; RNA-Seq
Cardiac tissues from LVAD cores at the time of LVAD implant (U/RR-pre) and adjacent to core samples at the time of explant (U/RR-post) from paired patient were flash frozen using liquid nitrogen. Identical regions from the apex of LV from explanted donors were used. Single nuclei suspensions were generated as previously described. In brief: flash frozen sections were minced with a razor blade, transferred to a Dounce Homogenizer containing 1 mL of lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% NP-40 in nuclease-free water) on ice. Samples were homogenized using five strokes, an additional 1 mL of lysis buffer added, and incubated on ice for 15 mins. Samples were then filtered with a 40μm filter and filter was rinsed with 1mL of lysis buffer. The mixture was then centrifuged at 500g for 5 min 4 °C, resuspended in 1mL nuclei wash buffer (2% BSA and 0.2 U μl−1 RNase inhibitor (Thermo Fisher, cat. no. AM2694) in 1× PBS) and, filtered using a 20m pluristrainer (Pluriselect, cat. No. SKU43-50020-03). Filtered solution as centrifuged using the above criteria and resuspended in 300 μL Nuclei Wash Buffer and transferred into a 5mL tube for flow cytometry. Subsequently, 1 μl DRAQ5 (5 mM solution; Thermo Fisher, cat. no. 62251) was added, sample gently vortexed, and allowed to incubate for 5 min prior to sorting. DRAQ5+ nuclei were sorted into 300 μL Nuclei Wash Buffer using a BD FACS Melody (BD Biosciences) with a 100 µM nozzle. Sorted nuclei were then centrifuged using the above conditions and resuspended in Nuclei Wash Buffer for a final target concentration of 1,000 nuclei/μL – nuclei were counted on a hemocytometer. Based on the nuclei concentration, 10,000 target nuclei were loaded onto a Chip G for GEM generation using the Chromium Single Cell 5ʹ Reagent v1.1 kit from 10X Genomics. Reverse transcription, barcoding, complementary DNA amplification and purification for library preparation were performed as per the Chromium 5ʹ v1.1 protocol at the McDonnel Genome Institute. Sequencing was performed on a NovaSeq 6000 platform (Illumina) at a target read depth of 100,000 at the McDonnel Genome Institute. Chromium Single Cell 5ʹ Reagent v1.1 kit from 10X Genomics