Illumina NovaSeq 6000 sequencing; Single Nuclei Sequencing of old and young mice hearts under senolytica (Ageing impairs the neuro-vascular interface in the heart)
Single Nuclei Sequencing of old and young mice hearts under senolytica (Ageing impairs the neuro-vascular interface in the heart)
To eliminate senescent cells from aged mice, a combination of the two senolytics drugs dasatinib and quercetin was used as proposed by Xu et al.(37). In brief, 5 mg/kg dasatinib (SML2589-50MG; Merck) and 50 mg/kg quercetin (Q4951-10G, Sigma-Aldrich) were applied via oral gavage to aged mice (18 months) on three consecutive days, every second week for a total duration of two months. Young (3 to 4 months) and aged (18 to 19 months) mice receiving the solvent (Phosal 50PG (368315, Lipoid) containing 3.3% ethanol (32221, Sigma-Aldrich) and 10% polyethylene glycol 400 (807485, Merck)) served as control cohorts. Cardiac function was monitored during the experiment using echocardiography and ECG traces as described below. The animal experiment has been conducted as approved by the state of Hessen. For Mouse hearts, the following protocol was used: cardiac tissues were thawed on ice and cut into small pieces. Minced tissue was pre-digested with a 5-ml enzyme solution of collagenase (2,500 U, Thermo Fisher Scientific) in HBSS+/+ (Gibco) for 10 min at 37 °C in a water bath. After centrifugation at 500g and 4 °C for 5 min, the supernatant was discarded, and nuclei were isolated after cell disruption with a glass dounce homogenizer (five strokes with a loose pestle and ten strokes with a tight pestle)40,41. After filtering (20-µm strainer, pluriSelect), the suspension was centrifuged at 1,000g and 4 °C for 6 min and resuspended in 500 µl staining buffer containing 1% BSA (Sigma-Aldrich), 5 nM MgCl2 (Sigma-Aldrich), 1 mM EDTA (Gibco), 1 mM EGTA (Gibco), 0.2 U µl−1 RNasin Plus Inhibitor (Promega) and 0.1 µg ml−1 Hoechst (Life Technologies) in Dulbecco´s phosphaste buffered saline (DPBS). Hoechst-positive nuclei were separated from cell debris by using the FACSAria Fusion instrument (BD Biosciences) and sorted into staining buffer without Hoechst at 4 °C. Nuclear suspensions were loaded on a 10x Chromium Controller (10x Genomics) according to the manufacturer's protocol. All snRNA-seq libraries were prepared using the Chromium Single Cell 3′ version 3 Reagent kit (10x Genomics) according to the manufacturer's protocol. Individual nuclei were isolated into droplets together with gel beads coated with unique primers bearing 10x cell barcodes, UMIs and poly(dT) sequences. Reverse transcription reactions generated barcoded full-length cDNA followed by disruption of emulsions using the recovery agent and cDNA cleanup with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific). Total cDNA was amplified using a Biometra Thermocycler TProfessional Basic Gradient with a 96-well sample block (98 °C for 3 min; cycled 14×: 98 °C for 15 s, 67 °C for 20 s and 72 °C for 1 min; 72 °C for 1 min; held at 4 °C). Amplified cDNA products were cleaned with the SPRIselect Reagent kit (Beckman Coulter). Indexed sequencing libraries were constructed using reagents from the Chromium Single Cell 3′ version 3 Reagent kit as follows: fragmentation, end repair and A tailing; size selection with SPRIselect; adaptor ligation; post-ligation cleanup with SPRIselect; sample index PCR and cleanup with SPRIselect beads. Library quantification and quality assessment were performed using the Bioanalyzer Agilent 2100 with a High Sensitivity DNA chip (Agilent Genomics). Indexed libraries were pooled equimolarly and sequenced on the Illumina NovaSeq 6000 by GenomeScan using paired-end 26 × 98-bp reads as the sequencing mode.