Illumina NovaSeq 6000 sequencing; Identification of cell-intrinsic effects of clonal hematopoiesis in heart failure
Identification of cell-intrinsic effects of clonal hematopoiesis in heart failure
Blood was obtained of patients with chronic heart failure at approximately the same time (between 10:00 AM and 12:00 PM) on study days. Informed consent was obtained from all patients. The study was approved by the local ethics review board and complies with the Declaration of Helsinki. Patients were eligible for inclusion into the study if they had stable chronic heart failure symptoms New York Heart Association (NYHA) classification of at least II, had a previous myocardial infarction at least 3 months before recruitment. Exclusion criteria were the presence of acutely decompensated heart failure with NYHA class IV, an acute ischemic event within 3 months prior to inclusion, a history of severe chronic diseases, documented cancer within the preceding 5 years, or unwillingness to participate. Blood was collected in sodium citrate–containing cell preparation tubes (CPT Vacutainer; Becton Dickinson) which were centrifuged at 1500xg for 20 minutes at room temperature. The upper layer containing mononuclear cells and plasma was collected and cells were washed and then stored until used for droplet scRNA-seq.  Cellular suspensions were loaded on a 10X Chromium Controller (10X Genomics) according to manufacturer's protocol based on the 10X Genomics proprietary technology. Single-cell RNA-Seq libraries were prepared using Chromium Single Cell 3′ Reagent Kit, v3 (10X Genomics) according to manufacturer's protocol. Briefly, the initial step consisted in performing an emulsion capture where individual cells were isolated into droplets together with gel beads coated with unique primers bearing 10X cell barcodes, UMI (unique molecular identifiers) and poly(dT) sequences.   Reverse transcription reactions were engaged to generate barcoded full-length cDNA followed by the disruption of emulsions using the recovery agent and cDNA clean up with DynaBeads MyOne Silane Beads (ThermoFisher). Bulk cDNA was amplified using a Biometra Thermocycler TProfessional Basic Gradient with 96-Well Sample Block (98°C for 3 minutes; cycled 14×: 98°C for 15s, 67°C for 20s, and 72°C for 1 minute; 72°C for 1 minute; held at 4°C). Amplified cDNA product was cleaned with the SPRIselect Reagent Kit (Beckman Coulter).