Single nuclear RNA sequencing from human endomyocardial biopsy (IVIG / Placebo treated) - raw/feature barcode matrix

Project description: See related publication Code repository of the related publication: https://github.com/fpeisker303/IVIG_snRNA_project/ Methods use to generate the Single nuclear RNA sequencing data Endomyocardial biopsies (EMB) were taken from the right ventricular septum and collected via the internal jugular vein using a transcatheter bioptome (Cordis, Miami, FL., USA) at baseline before the IVIg treatment and at the standardized six-months follow-up timepoint of the original study (i.e., median 6.4 [5.9-7.3] months). EMB were evaluated regarding viral persistent and immunohistology markers of inflammation and fibrosis. Spare cardiac biopsies were stored at -80°C until preparation of snRNA sequencing. The isolation of cardiac nuclei and the 10x library preparation were performed at the Max Delbrück Center for Molecular Medicine following a published protocol (1) with adaptations to low-sized tissue pieces (2). In brief, 1–4-mg-sized flash-frozen cardiac biopsies were placed in a pre-cooled dish and an equally sized droplet of homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl, 1 μM DTT, 1× protease inhibitor, 0.4 U μl−1 RNaseIn, 0.2 U μl−1 SUPERaseIn and 0.1% Triton X-100 in nuclease-free water) was added. Buffer-encapsulated tissue pieces were sliced with a scalpel. The tissue pieces were then transferred to a 7-ml glass Dounce tissue grinder (Merck), and nuclei were isolated and stained with NucBlue Live ReadyProbes Reagent (Thermo Fisher Scientific). Hoechst+ single nuclei were sorted via fluorescence-activated cell sorting (FACS) (BD Biosciences, FACSAria Fusion). Purity and integrity of nuclei were confirmed microscopically, and nuclei numbers were counted using a Countess II (Life Technologies) before processing with the Chromium Controller (10x Genomics) per the manufacturer’s protocol. Single-nucleus 3′ gene expression libraries were created using version 3.1 Chromium Single Cell Reagent Kits (10x Genomics) following the manufacturer's instructions. cDNA library quality control was performed using Bioanalyzer High Sensitivity DNA Analysis (Agilent Technologies) and a KAPA Library Quantification Kit. cDNA libraries were sequenced on an Illumina NovaSeq with a targeted read number of 30,000–50,000 reads per nucleus. Fastq files with sequencing results were processed using cellranger version 6.1.2 with the GRCh38-2020-A reference provided by 10x Genomics. References 1. Nadelmann ER, Gorham JM, Reichart D, Delaughter DM, Wakimoto H, Lindberg EL, et al. Isolation of Nuclei from Mammalian Cells and Tissues for Single-Nucleus Molecular Profiling. Curr Protoc. 2021;1(5):e132. 2. Maatz H, Lindberg EL, Adami E, López-Anguita N, Perdomo-Sabogal A, Cocera Ortega L, et al. The cellular and molecular cardiac tissue responses in human inflammatory cardiomyopathies after SARS-CoV-2 infection and COVID-19 vaccination. Nat Cardiovasc Res. 2025;4(3):330-45.