GSM7843074: WT_H, snRNAseq; Sus scrofa; RNA-Seq
Frozen heart tissue from the left ventricular free wall was minced with a blade and transferred to a Dounce homogenizer containing 500 µl of chilled lysis buffer. Samples were gently homogenized five to ten times without rotation, then incubated on ice for 5 minutes. A 700 µl lysis buffer was added to the lysate and filtered gently through a 70 µm filter into a 50 ml centrifuge tube. Then 1 ml 50% of iodixanol was added to the filtrate and mixed gently. A total of 2 ml of the nuclei suspension was transferred into a 15 ml centrifuge tube containing 3 ml of chilled iodixanol gradient solution (1 ml 33% iodixanol, 2 ml 30% iodixanol). Nuclei were recovered after centrifugation and resuspended gently in wash buffer, followed by counting on a hemocytometer. Nuclei were processed with the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 from 10× Genomics. In brief, cellular suspensions were loaded on a 10× Genomics GemCode Single-cell instrument to generate single-cell Gel Bead-In-EMlusion (GEMs). After dissolution of the Gel Bead in a GEM, reverse transcription, barcoding, complementary DNA amplification and purification for sequencing libraries preparation was performed according to the Single Cell 3' protocol.