Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of murine hearts after acute proteasome inhibition
Single cell RNA sequencing of murine hearts after acute proteasome inhibition
BALB/c mice were injected with CFZ (16 mg/kg, i.v.), CFZ and atRA (CFZ: 16 mg/kg i.v., atRA: 1 mg/kg i.v.) or Captisol (vehicle, i.v.) (2 mice per cohort). ECGs were recorded under anesthesia at the end of 1h treatment and the mice were sacrificed by cervical dislocation. Euthanized animals were perfused with 20 ml of PBS and small heart tissue pieces were digested three times for 20 min at 37°C with 120 µg/ml collagenase P (Roche), 25 µg/ml DNase I (Applichem, Germany) in medium (RPMI-1640 containing 5% FCS (Sigma-Aldrich, Switzerland) and10 mM HEPES (Sigma-Aldrich, Switzerland). To eliminate remaining erythrocytes, the cell suspensions were incubated with MACS anti-Ter119 microbeads (Miltenyi Biotec, Germany) and passed through a MACS LS column (Miltenyi Biotec, Germany). Unbound single cell suspensions were stained with Live/Dead Fixable e780 Aqua staining. Live cells were sorted with a BD FACS Melody cell sorter (BD Biosciences, CA, USA). cDNA library generation was performed following the established commercial protocol for Chromium Single Cell 3' Reagent Kit (v3 Chemistry).