title: GSM4462453: Mouse, adult, male, knockout 76; Mus musculus; RNA-Seq
library_construction_protocol: The left kidney was excised, decapsulated, cut into 5 mm cubes and snap frozen. Nuclei were isolated with Nuclei EZ Lysis buffer (NUC-101; Sigma-Aldrich) supplemented with protease inhibitor (cOmplete™ ULTRA tab; Sigma-Aldrich) and RNase inhibitors: RNAsin Plus (5 ml/ml, Promega, Madison, WI) and SUPERaseIN (5 ml/ml , ThermoFisher The frozen kidney cubes were placed in a sterile petri dish with 1 ml of Nuclei EZ lysis buffer (plus protease and RNase inhibitors) and quickly minced.  The tissue was then homogenized with RNase-free, disposable pellet pestles and tubes (Fisher #12-141-368), and a hand held, Bel-Art™ Micro-Tube homogenizer (Wayne, NJ).  The homogenate was filtered through a 200 micron strainer (all strainers were purchase from pluriSelect Life Science), and homogenized again with a fresh tube and pestle.  The homogenate was moved to a sterile, RNase-free 15 ml tube with an additional 2 ml of Nuclei EZ lysis buffer (plus protease and RNase inhibitors).  After a 5 min incubation on ice, the homogenate was filtered through a 40 micron strainer.  The subsequent homogenate was centrifuged at 500 g for 5 min at 4°C.  The pellet was resuspending in Nuclei EZ lysis buffer (plus 1 ml/ml of SUPERaseIN and RNAsin), incubated on ice for 5 min and centrifuged again.  The pellet was resuspended in 2 ml of 1X Dulbecco's Phosphate-buffered saline + 1% bovine serum albumin, filtered through a 5 micron strainer, and nuclei immediately counted with a hemocytometer. At least 5000 nuclei from each sample were placed into oil droplets with the 10X Genomics Chormium™ Single Cell B Chip kit, and cDNA libraries made using 10x Chorminum™ Single cell 3' library and gel bead kit (version 3, 10X Genomics). cDNA libraries made using 10x Chorminum™ Single cell 3' library and gel bead kit (version 3, 10X Genomics).
attributes.tag: GEO Accession
attributes.value: GSM4462453