GSM9307789: P2; Sus scrofa; RNA-Seq
Nuclei were isolated as previously described [35] with minor modifications. Briefly, 400 mg of frozen myocardial tissue per sample was thawed on ice, rinsed with cold phosphate-buffered saline (PBS), and minced into small pieces in 1 mL lysis buffer (composition: 0.32 M sucrose, 1.5 mM CaCl₂, 0.9 mM magnesium acetate (C₄H₆MgO₄), 0.75 mM ethylene glycol tetraacetic acid (EGTA), 3 mM Tris-HCl (pH 8.0), 0.6 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 0.3 mM dithiothreitol (DTT), and 20 U/μL RNase inhibitor (RI)). Next, the tissue was homogenized in 15 mL lysis buffer using a T-25 Ultra-Turrax probe homogenizer (IKA, Staufen, Germany) at 24,000 rpm for 20 seconds, then diluted to 30 mL with additional lysis buffer. Homogenates were further disrupted using a 40 mL glass dounce homogenizer (10 strokes) to release nuclei, then filtered sequentially through 100 μm and 70 μm cell strainers. Filtrates were centrifuged at 700 × g for 10 minutes at 4 °C; pellets were resuspended in 23 mL sucrose buffer (2.1 M sucrose, 0.9 mM magnesium acetate, 3 mM Tris-HCl (pH 8.0), 0.2 mM DTT, 20 U/μL RI). A 7 mL sucrose buffer cushion (pre-incubated with 1% bovine serum albumin [BSA]) was layered in a 50 mL centrifuge bottle (Beckman Coulter cat no. 357003), over which 23 mL of the resuspended pellet was carefully layered. The bottle was centrifuged at 13,000 rpm for 1 hour at 4 °C (Beckman Avanti J-26XPI centrifuge). Supernatants were discarded, and nuclei pellets resuspended in 1 mL 1% BSA (containing 200 U/mL RI). Nuclei were counted using trypan blue staining, adjusted to 700–1000 nuclei/μL, and processed for single-nucleus RNA sequencing (snRNA-seq) following the 10x Genomics protocol. Isolated nuclei were stained with acridine orange/propidium iodide (AO/PI) and counted using a fluorescence-based automated cell counter. Nuclei concentration was adjusted to 800–1200 nuclei/μL and ~20,000 nuclei per sample were loaded onto a Chromium Controller (10x Genomics) for gel bead emulsion formation. Libraries were prepared using the Chromium Next GEM Single Cell 3′ GEM, Library & Gel Bead Kit v3.1 (10x Genomics cat. no. PN-1000121) according to the manufacturer's instructions. Libraries were sequenced on the Illumina PE150 platform at a depth of ~40,000 reads per nucleus.  All samples' libraries were within the range of 200-2000 bp. No additional peaks were observed below 200 bp, ensuring that the libraries were qualified.