title: Endothelial expression of ZBTB16 protects against cardiac aging
design: Endothelial expression of ZBTB16 protects against cardiac aging
library_construction_protocol: To isolate nuclei for transcriptomic analysis, hearts were perfused with 1X DPBS in vivo, snap-frozen in liquid nitrogen and stored at -80°C until usage. Eight mice hearts were sequenced (n=4 per group, two females and two males, respectively). For the nuclei isolation, one female and one male heart per group were pooled and processed together. The hearts were minced in pre-filtered homogenization buffer containing 250 mM sucrose (1623637, Sigma-Aldrich), 25 mM KCl (AM9640G, Invitrogen), 5 mM MgCl2 (AM9530G, Invitrogen), 10 mM Tris buffer pH 8.0 (AM9855G, Invitrogen), 1 mM DTT (P2325, Thermo-Fisher Scientific), 1X protease inhibitor (11697498001, Roche), 0.6 U/µL Ambion RNase inhibitor (AM2682, Thermo-Fisher Scientific), 0.1% Triton (T8787, Sigma-Aldrich) and 2% BSA (A8022, Sigma-Aldrich) in ultra-pure DNase/RNase-free distilled water (10977035, Thermo-Fisher Scientific). Cells were disrupted with seven strokes of a loose pestle in a glass dounce homogenizer. The homogenized solution was filtered using a pre-wetted 40 µm cell strainer followed by a pre-wetted 20 µm cell strainer. After centrifugation (500 g at 4°C for 8 minutes), the cell pellet was re-suspended in sorting buffer containing 2% BSA, 0.6 U/µL Ambion RNase inhibitor, 1 mM DTT in DPBS. 7AAD (420403, Biolegend) positive nuclei were separated from cell debris using the FACS-Aria III instrument (BD Biosciences; Nozzle size 100 microns) into collection buffer containing 2% BSA, 1 U/µL Ambion RNase inhibitor, 1 mM DTT in DPBS. Sorted nuclei were washed with 0.04% BSA in DPBS. Nuclei suspensions were loaded on a 10X Chromium Controller (10X Genomics) according to manufacturer's protocol based on the 10X Genomics proprietary technology. All snRNA-seq libraries were prepared using Chromium Single Cell 3′ v3 Reagent Kit (10X Genomics) according to manufacturer's protocol. Briefly, the initial step consisted in performing an emulsion where individual nuclei were isolated into droplets together with gel beads coated with unique primers bearing 10X cell bar-codes, 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 (Thermo Fisher Scientific). Total cDNA was amplified using a Biometra Thermocycler TProfessional Basic Gradient with 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 product was cleaned with the SPRIselect Reagent Kit (Beckman Coulter). Indexed sequencing libraries were constructed using the reagents from the Chromium Single Cell 3′ v3 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 SPRI select beads. 