GSM9461259: Cardiac nuclei, Sham surgery, NS treatment, replicate 1; Mus musculus; RNA-Seq
Nuclei were isolated from flash-frozen and powdered heart tissue. A detergent-mechanical cell lysis method was used, involving three major steps: lysis, homogenization, and density barrier centrifugation. In a laminar hood, a ~50-mg powered heart tissue was placed into a pre-frozen BioPulverizer (BioSpec), then immediately added lysis buffer (10mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1 Nonidet P40, 1 X RNase inhibitor, 1ul/mL DEPC). Tissue was disaggregated, flushing up and down, first using a 1 ml pipette tip. Homogenized tissue was diluted to 2 ml with lysis buffer and filtered through a 70 µm filter. Homogenate was spun at 350 g for 5 min at 4°C. The pellet was washed twice by washing buffer (1 X PBS, 1% BSA, 1 X RNase inhibitor). Pellet was then resuspended and 500 µl of pellet suspension was diluted with 500 µl 5% iodixanol solution (5% iodixanol, 250 mM sucrose, 150 mM KCl, 130 mM MgCl2, 1 X RNase inhibitor, and 60mM Tris-HCl, pH 8.0), filtered for the second time with a 20 µm mesh and placed on bottom of 500 µl layer of 8.5% iodixanol solution (8.5% iodixanol, 250 mM sucrose, 150 mM KCl, 130 mM MgCl2, 1 X RNase inhibitor, 60mM Tris-HCl, pH 8.0). The density barrier was centrifuged at 10,000 g for 10 minutes at 4°C. The pellet was collected and washed with washing buffer, centrifuged at 350 g for 5 min at 4°C, and finally suspended in 50 µl washing buffer. Single nuclei suspensions were counted and evaluated for integrity using Propidium Iodide in a MoxiGo cytometer using 650 nm filter. The nuclei count was adjusted to 1000 nuclei/ml. Single-cell RNA-seq libraries were prepared using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index) (10x Genomics, Pleasanton, CA, USA), following the manufacturer's user guide (CG000315 Rev C). Briefly, single cells were encapsulated into Gel Beads-in-Emulsion (GEMs) using the Chromium Controller and a microfluidic Chip G. Each GEM contains a single cell, a uniquely barcoded gel bead, reverse transcription reagents, and partitioning oil. Within each GEM, polyadenylated mRNAs were reverse transcribed to generate barcoded full-length cDNA, incorporating a 10x Barcode and Unique Molecular Identifier (UMI) through oligo(dT) primers. After reverse transcription, GEMs were broken, and barcoded cDNA was purified using Silane DynaBeads. The purified cDNA was PCR amplified and size-selected using SPRIselect magnetic beads. For library construction, cDNA was enzymatically fragmented, end-repaired, A-tailed, and ligated with Illumina-compatible adaptors. Dual indices (i7 and i5) were incorporated during sample index PCR. Final libraries included P5 and P7 adapter sequences and were compatible with Illumina sequencing. Libraries were quantified and assessed for quality using an Agilent Bioanalyzer, TapeStation, or equivalent platform, and sequenced on an Illumina instrument using paired-end mode (e.g., 28 bp for Read 1, 8 bp i7 index, 10 bp i5 index, and 90 bp for Read 2).