GSM5928091: Substantia nigra, Tg-Mlkl-/-; Mus musculus; RNA-Seq
All procedures were carried out on the ice or at 4 °C. Briefly, the frozen substantia nigra brain tissue was homogenized in 2 ml Homogenization Buffer (10 mM Tris-HCl, pH 8.0, 25 mM KCl, 5 mM MgCl2, 0.1% Triton X-100, 250 mM sucrose, 0.5 U/μl SuperRNase inhibitor, and 0.5 U/μl RNAse inhibitor), using a Wheaton Dounce Tissue Grinder (10 strokes with the loose pestle and 15 strokes with the tight pestle). Then, tissue homogenate was overlaid onto 10% iodixanol and centrifuged with an SW32 rotor at 10,000g for 20 minutes at 4 °C. After ultracentrifugation, the supernatant was aspirated and discarded, and the remaining pellet was resuspended in 0.5 % BSA plus 50 ng/ul DAPI for nuclei isolation using a BD AriaFusion cell sorter (BD Biosciences). RNA libraries were prepared for sequencing using Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3, 16 rxns PN-1000075 Single nuclei RNA-Seq