GSM9432994: Mouse_heart_TAC_Sham_5D_Chip3; Mus musculus; RNA-Seq
Cardiac cells were isolated from whole hearts using a modified Langendorff perfusion protocol. Hearts were perfused with a buffer containing 150 mM NaCl, 5 mM HEPES, 5.4 mM KCl, 10 mM glucose, 2 mM Na-pyruvate, 1.2 mM MgCl2, 10 mM taurine, and 12.38 mM 2,3-butanedione monoxime for 2 min at 37°C, followed by perfusion with the same buffer supplemented with 25 mM CaCl2 and 210 µg/mL Liberase DH (Roche) for 10 min at 37°C. Reactions were stopped with buffer containing 0.5% w/v bovine serum albumin and 200 µM CaCl2. Hearts were minced and homogenized into a single cell suspension. Samples processed using ICELL8 System (Takara Bio). Cells distributed on 5,184 nanowell ICELL8 250v chips. Single alive cells identified using Hoechst 33342 and propidium iodide staining with CellSelect Software. cDNA synthesis performed by oligo-dT priming in one-step RT-PCR. P5 indexing primers, Terra Polymerase added for library prep. Transposase enzyme (Tn5 mixture) added to each well. P7 indexing primers dispensed. Final libraries amplified, pooled, and purified with AMPureXP beads (Beckman Coulter) with size selection to obtain ~500bp average library size.