GSM8827076: single-nucleus RNA-seq, postnatal day 14, replicate 2; Mus musculus; RNA-Seq
Hearts were harvested from mice at postnatal day 0 (p0, 2 replicates), day 7 (p7, 2 replicates), day 14 (p14, 2 replicates), and day 21 (p21, 2 replicates), respectively. Hearts were perfused with KCl and PBS before harvesting. After removing the heart from the chest, hearts were minced into pieces on a glass petri dish on ice using a razor blade. Minced heart tissue was then transferred to a 15 ml falcon tube for tissue homogenization with 1 ml of homogenization buffer (0.32M sucrose, 1mM Tris-HCl PH8.0, 3mM Magnesium Acetate, 0.1mM EDTA, 5mM Calcium Chloride, 0.1% Triton, 1x cOmplete Protease Inhibitor Cocktail, 200 U/ml RNase OUT, 1mM DTT). The tissue homogenization was performed on ice with two cycles of 30 second on and 60 sec off. After the tissue was completely homogenized, the tissue lysate was then transferred to a 7 ml tissue grinder. 1 ml of tissue homogenization buffer was used to wash the falcon tube before adding it to the tissue grinder. Eight strokes were performed with a loose pestle (A) on ice. 30 strokes were performed with a tight pestle (B) on ice. Then the tissue lysate was passed through a 70 μm and followed with a 40 μm cell strainer. Wash off any left-over tissue lysate from the 40 μm cell strainer with 1 ml of the homogenization buffer. Then the nuclei were pelleted by centrifuging at 500 g for 5 min at 4°C. Wash the pellet twice with resuspension buffer (PBS/1%BSA, 200 U/ml RNase OUT, 1 mM DTT). The nuclei pellets were then resuspended in 0.5 ml resuspension buffer. Before running the nuclei pellet with a 10x 3' scRNA-seq kit, isolated nuclei were labeled with DRAQ5 and sorted with flow cytometry to ensure the purity of the nuclei. The single-nucleus library preparations were performed strictly following the manufacturer's manual (Chromium Single Cell 3' Reagent Kits User Guide, CG000315).