GSM5610192: AR1_P56_8096CZ; Sus scrofa; RNA-Seq
GSM5610192
Tissues were cut while submerged in cold phosphate-buffered saline (PBS) or UW  solution and washed to remove blood; then, the solution was replaced with 1 mL lysis buffer (10  mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% Nonidet™ P40 Substitute, and 50 U/mL RNase  inhibitor in DEPC-treated water). Tissues were cut into even smaller pieces and aspirated with  the lysis buffer into a 50 mL tube; then, 10 mL of lysis buffer was added, the tissues were  ground for 20-30 s, and more lysis buffer was added. The mixture was placed on ice for ten minutes, filtered with 100-μm and 70-μm strainers, and centrifuged for 5 min at 700 rcf and 4°C; then, the supernatant was removed, and the pellet was resuspended in 10 mL nuclei wash and resuspension buffer (1X PBS, 1.0% bovine serum albumin [BSA], and 50 U/mL RNase  inhibitor). The suspension was passed through a 40-μm strainer and the nuclei were centrifuged again for 5 min at 700 rcf and 4°C. The supernatant was removed and the pellet was resuspended in 1 mL nuclei wash and resuspension buffer; then, the nuclei were centrifuged again for 5 min at 700 rcf and 4 °C, the supernatant was removed, and the pellet was resuspended in 5 mL sucrose cushion buffer I (2.7 mL Nuclei PURE 2M Sucrose Cushion  Solution and 300 μL Nuclei PURE Sucrose Cushion Buffer) and mixed with 10 mL sucrose  buffer. The mixture was layered over 5 mL of sucrose cushion buffer in a second Eppendorf  tube and then centrifuged for 60 min at 13000 g and 4 °C. All but 100 μL of the supernatant was removed, and nuclei wash and resuspension buffer was added. The nuclei were resuspended, and the solution was passed through a 40-μm strainer; then, the nuclei concentration was determined with a cell counter or hemocytometer and adjusted to 1000 nuclei/μL. The nuclei  were placed on ice, stained with propidium iodide for 5 min, and then immediately processed via the 10× Genomics® Single-Cell Protocol.