GSM7065665: WT_P_snRNAseq; Sus scrofa; RNA-Seq
One pancreatic tissue piece (approximately 0.2 g) was taken from each group of pigs from a similar location. The tissue was chopped into 1-2 cubic millimeter size pieces on ice, and the pieces were immediately frozen in liquid nitrogen for 5 min, and then transferred to a Dounce homogenizer and lysis buffer (0.25 M sucrose, 5 mM CaCl2, 3 mM MgAc2, 10 mM Tris-HCL PH 8.0, 1 mM DTT, 0.1 mM EDTA, 1×Protease Inhibitor, 1 U, ul Ribolock RNase Inhibitor) was added to submerge the tissue. Press the tissue vertically 15-30 times by applying a grinding rod to turn the tissue into a homogenate. Add Nuclear Wash Buffer (0.04% BSA, 0.2 U, ul Ribolock RNase Inhibitor, 500 mM mannitol, 0.1 mM PMSF Protease Inhibitor in PBS) and mix upside down. The filtrate was obtained by filtering the tissue homogenate using a cell strainer(70um). The tissue homogenate was filtered using a cell strainer (70um) and 50%, 33%, and 30% iodixanol (0.16M sucrose, 10mM NaCl2, 3mM MgCl2, 10mM Tris-HCl PH 7.4, 1 U, ul Ribolock RNase Inhibitor, 0.1 mM DTT, 0.1mM PMSF Protease Inhibitor) was added slowly to the filtrate in sequence. Centrifuge at 4°C, 10,000×g for 20 min. After removing the top iodixanol, the nuclei were collected at the 30% iodixanol interface, resuspended by Nuclear Wash Buffer, centrifuged at 4°C, 500×g for 5 min, and the upper supernatant was aspirated and filtered through a 40um cell strainer to remove cell debris and large nuclei masses. Nuclear concentrations were manually assessed using 0.4% trypan blue counterstaining and hemocytometry. Finally, nuclear concentrations were adjusted to 700-1200 nuclei, µl. R1 (read 1 primer sequence) were added to the molecules during GEM incubation. P5, P7, a sample index, and R2 (read 2 primer sequence) were added during library construction via End Repair, A_x0002_tailing, Adaptor Ligation, and PCR. The final libraries contained the P5 and P7 primers used in Illumina bridge amplification.