GSM8459467: SC10_1 - DKOData_DKOData (650); Mus musculus; RNA-Seq
Kidneys were perfused with HBSS and stored in RNALater at -80C. A 2 mm3 section of tissue was rinsed briefly with PBS and minced before adding 1 mL of lysis buffer containing 20 mM Tris-HCl (pH 8), 320 mM sucrose, 5 mM CaCl2, 3 mM MgAc2,  0.1 mM EDTA, and 0.1% TritonX-100. The tissue was initially dissociated by pipetting up and down ~10X with a p-1000 tip and then being passed through a 25G syringe ~10X. The tissue was incubated on ice for 10 minutes and then passed through a 30 µm CellTrics filter. Nuclei were pelleted by centrifugation (5 min, 500g, 4°C) and washed with PBS after removal of supernatant. Nuclei were pelleted again and resuspended in 1 mL of PBS containing 0.04% BSA and RNAse inhibitor (0.2u/µL) before counting by hemocytometer.  Nuclei were then diluted and prepared using the 10X Chromium System according to the manufacturer's instructions (10X Genomics). RNA libraries were prepared for sequencing using standard 10X Genomics and Illumina protocols