title: GSM7794811: Pig, Perfused, Replicate 1; Sus scrofa; RNA-Seq
library_construction_protocol: A snap frozen kidney tissue sample from pig, mouse, or rat was thawed on ice and 3-4 mm of the core was chopped with a razor blade in a petri dish on ice and homogenized using a Dounce homogenizer (D8938-1 SET, Sigma-Aldrich) in 200 µl ice-cold lysis solution and incubated on ice for 20-30 min with additional 3.8 ml of ice-cold lysis solution. Lysis solution was prepared with Nuclei PURE lysis buffer (NUC-201, Sigma-Aldrich), 1 mM dithiothreitol (D9779, Sigma-Aldrich) and 0.1 % Triton X-100 (NUC-201, Sigma-Aldrich) according to manufacturer protocol and an RNAse inhibitor mix (0.04 U/µl SUPERaseIN RNAse Inhibitor [AM 2696, Thermo Fisher]; 0.04 U/µl RNAsin Plus RNAse Inhibitor [N2615, Promega]) was added. The single nuclei suspension was filtered through a 30 µm strainer (04-004-2326, Sysmex) and centrifuged at 500g for 5 min at 4°C. The pellet obtained from dissociated rat kidney tissue was resuspended in 2% BSA in DPBS with 0.04 U/µl SUPERaseIN RNAse Inhibitor and 0.04 U/µl RNAsin Plus RNAse Inhibitor and nuclei were counted. The pellet obtained from dissociated mouse and pig kidney tissue was resuspended and incubated for 2 min in 1ml red blood cell lysis buffer hybri-max™ (R7757-100ml, Sigma Aldrich), filtered through a 5 µm strainer (04-004-2323, Sysmex) and washed with 4 ml of ice-cold 0,01% BSA (AM2616, Thermo Fisher) in DPBS (59331C; Sigma) 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 (mouse and pig) in DPBS with 0.04 U/µl SUPERaseIN RNAse Inhibitor and 0.04 U/µl RNAsin Plus RNAse Inhibitor and nuclei were counted. Nuclei number was counted and samples were diluted before loading to the Chromium 10X device to capture ~ 10 000 nuclei. 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.