GSM7665186: C2; Sus scrofa; RNA-Seq
Lysis solution was prepared with Nuclei PURE lysis buffer, 1 mM dithiothreitol and 0.1 % Triton X-100 according to manufacturer protocol and an RNAse inhibitor mix was added. The single nuclei suspension was filtered through a 30 µm strainer  and centrifuged at 500g for 5 min at 4 °C. The pellet was resuspended and incubated for 2 min in 1ml red blood cell lysis buffer hybri-max™ , filtered through a 5 µm strainer and washed with 4 ml of ice -cold 0,01% BSA in DPBS with 0.04 U/µl SUPERaseIN RNAse Inhibitor and 0.04 U/µl RNAsin Plus RNAse Inhibitor at 500 g for 5 min at 4 °C. The pellet was resuspended in 1% BSA in DPBS with 0.04 U/µl SUPERaseIN RNAse Inhibitor and 0.04 U/µl RNAsin Plus RNAse Inhibitor. Nuclei number was counted and samples were diluted prior to loading to the Chromium 10X device to capture ~ 6,000 nuclei of one snap frozen, one RNAlater and the CellCover stored sample and to capture ~ 10,000 nuclei of two snap frozen and one RNAlater stored sample The nuclei were separated into Gel Bead Emulsion droplets and libraries were prepared with the Chromium NEXT GEM Single Cell 3' Reagent kits v3.1 according to manufacturer's protocol. The libraries were sequenced on an Illumina Novaseq6000 platform as symmetric paired end runs (150 bases) with 200 million raw sequencing reads per sample (pig)