GSM8514648: Heart, running 16 weeks; Mus musculus; RNA-Seq
Two (baseline) and four (infarct) mouse hearts from male and female mice were pooled together per group for further processing. Cardiac ventricles were collected, snap frozen in liquid-N2 and stored at -80°C until further processing. Hearts were allowed to sit on ice in a petri dish for 5 min, before mincing them with surgical blades (Fine Science Tools, #10020-00) into a paste-like mash. The minced sample was moved to a 15 ml Dounce homogenizer together with 5 ml of ice-cold lysis buffer (NaCl 146 mM, Tris-HCl 10 mM, CaCl2 1 mM, MgCl2 21 mM, 0.5% CHAPS, 0.1% BSA, SUPERnase InTM RNase inhibitor 20 U/ml [AM2696, ThermoFisher Scientific]) and left to lyse for another 5 min on ice. Then, 9 ml of wash buffer (2% BSA in PBS with 20 U/ml SUPERnase InTM RNase inhibitor) was added and the pestle A was lowered 10 times to homogenize the tissue. The resulting suspension was filtered first through 70 µm strainer, then through 40 µm strainer  and subsequently transferred to a clean 15 ml centrifuge tube. Following a centrifugation (450 g for 5 minutes at 4°C), the supernatant was removed, and the pellet was washed with 10 ml of wash buffer and centrifuged again. After the supernatant was removed, the nuclei-containing pellet was transferred to a 5 ml round bottom FACS tube and stained with Hoechst 34580 (Thermo Fisher Scientific, #H21486) in PBS for 5 minutes. Hoechst+ nuclei were sorted (85 µm nozzle, 4-way purity sorting mode) using a BD FACSAria III into the wash buffer. and immediately loaded into the 10x Genomics Chromium instrument. Nuclei obtained using FACS were suspended in wash buffer targeting the required a concentration of 1,000 cells/μl and loaded onto the 10x Genomics Chromium controller (Next GEM Chip G, #2000177). Samples were loaded into 4 wells, corresponding to one well per experimental group (sedentary, running, sedentary-infarct, running-infarct). Libraries were generated following 10x Genomics Chromium Next GEM Single Cell 3' Reagent Kits v3.1 Dual Index (CG000315 Rev E). Quality control of cDNA and final libraries was done using 4200 TapeStation System (Agilent) and D5000 High Sensitivity ScreenTape and Reagents. Libraries were sequenced on the Illumina Novaseq 6000 system.